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Sample records for eukaryotic genes derived

  1. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular.

  2. Horizontal gene transfer in eukaryotes: the weak-link model.

    PubMed

    Huang, Jinling

    2013-10-01

    The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes.

  3. Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes.

    PubMed

    Yabuki, Akinori; Toyofuku, Takashi; Takishita, Kiyotaka

    2014-07-01

    Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.

  4. Gene Positioning Effects on Expression in Eukaryotes.

    PubMed

    Nguyen, Huy Q; Bosco, Giovanni

    2015-01-01

    The packaging and organization of the genome within the eukaryotic interphase nucleus directly influence how the genes are expressed. An underappreciated aspect of genome structure is that it is highly dynamic and that the physical positioning of a gene can impart control over its transcriptional status. In this review, we assess the current knowledge of how gene positioning at different levels of genome organization can directly influence gene expression during interphase. The levels of organization discussed include chromatin looping, topologically associated domains, chromosome territories, and nuclear compartments. We discuss specific studies demonstrating that gene positioning is a dynamic and highly regulated feature of the eukaryotic genome that allows for the essential spatiotemporal regulation of genes.

  5. Endosymbiotic origin and differential loss of eukaryotic genes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Sousa, Filipa L; Lockhart, Peter J; Bryant, David; Hazkani-Covo, Einat; McInerney, James O; Landan, Giddy; Martin, William F

    2015-08-27

    Chloroplasts arose from cyanobacteria, mitochondria arose from proteobacteria. Both organelles have conserved their prokaryotic biochemistry, but their genomes are reduced, and most organelle proteins are encoded in the nucleus. Endosymbiotic theory posits that bacterial genes in eukaryotic genomes entered the eukaryotic lineage via organelle ancestors. It predicts episodic influx of prokaryotic genes into the eukaryotic lineage, with acquisition corresponding to endosymbiotic events. Eukaryotic genome sequences, however, increasingly implicate lateral gene transfer, both from prokaryotes to eukaryotes and among eukaryotes, as a source of gene content variation in eukaryotic genomes, which predicts continuous, lineage-specific acquisition of prokaryotic genes in divergent eukaryotic groups. Here we discriminate between these two alternatives by clustering and phylogenetic analysis of eukaryotic gene families having prokaryotic homologues. Our results indicate (1) that gene transfer from bacteria to eukaryotes is episodic, as revealed by gene distributions, and coincides with major evolutionary transitions at the origin of chloroplasts and mitochondria; (2) that gene inheritance in eukaryotes is vertical, as revealed by extensive topological comparison, sparse gene distributions stemming from differential loss; and (3) that continuous, lineage-specific lateral gene transfer, although it sometimes occurs, does not contribute to long-term gene content evolution in eukaryotic genomes.

  6. The Tangled Web: Gene Genealogies and the Origin of Eukaryotes.

    PubMed

    Katz

    1999-10-01

    Accessing data from the genomes of organisms (individual genes) and analyzing these data using sophisticated alignment and phylogenetic methods led to the expectation that we would be able to paint a clear picture of the evolution of eukaryotes. Previous analyses based on morphology and ultrastructure failed to pinpoint both the sister taxon to eukaryotes and the branching order of eukaryotic lineages. However, the expectation that molecular data would provide resolution has not been met since a growing number of gene genealogies present conflicting hypotheses for the origin and diversification of eukaryotes. Instead of reconstructing a simple bifurcating tree of life, these gene genealogies have generated a complex picture of eukaryotic genomes whereby ancient lateral transfers (of individual genes or perhaps even entire genomes) has tangled the evolutionary history of eukaryotes. Resolution of these conflicting genealogies comes in recognizing that eukaryotes are chimeric, containing genetic information from multiple ancestral lineages.

  7. Evolutionary Origins of the Eukaryotic Shikimate Pathway: Gene Fusions, Horizontal Gene Transfer, and Endosymbiotic Replacements†

    PubMed Central

    Richards, Thomas A.; Dacks, Joel B.; Campbell, Samantha A.; Blanchard, Jeffrey L.; Foster, Peter G.; McLeod, Rima; Roberts, Craig W.

    2006-01-01

    Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been

  8. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

  9. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation. PMID:25733873

  10. Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

    PubMed

    Chou, Seemay; Daugherty, Matthew D; Peterson, S Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L; Fritz-Laylin, Lillian K; Ferrin, Michael A; Harding, Brittany N; Jacobs-Wagner, Christine; Yang, X Frank; Vollmer, Waldemar; Malik, Harmit S; Mougous, Joseph D

    2015-02-01

    Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems. PMID:25470067

  11. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    PubMed Central

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic—and plant and algal—lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller’s ratchet—the origin of eukaryotic recombination, or sex—might have required surprisingly little evolutionary innovation. PMID:25733873

  12. Massive expansion of the calpain gene family in unicellular eukaryotes

    PubMed Central

    2012-01-01

    Background Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Results Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. Conclusions The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes. PMID:23020305

  13. Eukaryotic genes of archaebacterial origin are more important than the more numerous eubacterial genes, irrespective of function.

    PubMed

    Cotton, James A; McInerney, James O

    2010-10-01

    The traditional tree of life shows eukaryotes as a distinct lineage of living things, but many studies have suggested that the first eukaryotic cells were chimeric, descended from both Eubacteria (through the mitochondrion) and Archaebacteria. Eukaryote nuclei thus contain genes of both eubacterial and archaebacterial origins, and these genes have different functions within eukaryotic cells. Here we report that archaebacterium-derived genes are significantly more likely to be essential to yeast viability, are more highly expressed, and are significantly more highly connected and more central in the yeast protein interaction network. These findings hold irrespective of whether the genes have an informational or operational function, so that many features of eukaryotic genes with prokaryotic homologs can be explained by their origin, rather than their function. Taken together, our results show that genes of archaebacterial origin are in some senses more important to yeast metabolism than genes of eubacterial origin. This importance reflects these genes' origin as the ancestral nuclear component of the eukaryotic genome. PMID:20852068

  14. An Evolutionary Network of Genes Present in the Eukaryote Common Ancestor Polls Genomes on Eukaryotic and Mitochondrial Origin

    PubMed Central

    Thiergart, Thorsten; Landan, Giddy; Schenk, Marc; Dagan, Tal; Martin, William F.

    2012-01-01

    To test the predictions of competing and mutually exclusive hypotheses for the origin of eukaryotes, we identified from a sample of 27 sequenced eukaryotic and 994 sequenced prokaryotic genomes 571 genes that were present in the eukaryote common ancestor and that have homologues among eubacterial and archaebacterial genomes. Maximum-likelihood trees identified the prokaryotic genomes that most frequently contained genes branching as the sister to the eukaryotic nuclear homologues. Among the archaebacteria, euryarchaeote genomes most frequently harbored the sister to the eukaryotic nuclear gene, whereas among eubacteria, the α-proteobacteria were most frequently represented within the sister group. Only 3 genes out of 571 gave a 3-domain tree. Homologues from α-proteobacterial genomes that branched as the sister to nuclear genes were found more frequently in genomes of facultatively anaerobic members of the rhiozobiales and rhodospirilliales than in obligate intracellular ricketttsial parasites. Following α-proteobacteria, the most frequent eubacterial sister lineages were γ-proteobacteria, δ-proteobacteria, and firmicutes, which were also the prokaryote genomes least frequently found as monophyletic groups in our trees. Although all 22 higher prokaryotic taxa sampled (crenarchaeotes, γ-proteobacteria, spirochaetes, chlamydias, etc.) harbor genes that branch as the sister to homologues present in the eukaryotic common ancestor, that is not evidence of 22 different prokaryotic cells participating at eukaryote origins because prokaryotic “lineages” have laterally acquired genes for more than 1.5 billion years since eukaryote origins. The data underscore the archaebacterial (host) nature of the eukaryotic informational genes and the eubacterial (mitochondrial) nature of eukaryotic energy metabolism. The network linking genes of the eukaryote ancestor to contemporary homologues distributed across prokaryotic genomes elucidates eukaryote gene origins in a

  15. Mechanisms of gene targeting in higher eukaryotes.

    PubMed

    Tokunaga, Akinori; Anai, Hirofumi; Hanada, Katsuhiro

    2016-02-01

    Targeted genome modifications using techniques that alter the genomic information of interest have contributed to multiple studies in both basic and applied biology. Traditionally, in gene targeting, the target-site integration of a targeting vector by homologous recombination is used. However, this strategy has several technical problems. The first problem is the extremely low frequency of gene targeting, which makes obtaining recombinant clones an extremely labor intensive task. The second issue is the limited number of biomaterials to which gene targeting can be applied. Traditional gene targeting hardly occurs in most of the human adherent cell lines. However, a new approach using designer nucleases that can introduce site-specific double-strand breaks in genomic DNAs has increased the efficiency of gene targeting. This new method has also expanded the number of biomaterials to which gene targeting could be applied. Here, we summarize various strategies for target gene modification, including a comparison of traditional gene targeting with designer nucleases.

  16. Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts

    PubMed Central

    Marsit, Souhir; Mena, Adriana; Bigey, Frédéric; Sauvage, François-Xavier; Couloux, Arnaud; Guy, Julie; Legras, Jean-Luc; Barrio, Eladio; Dequin, Sylvie; Galeote, Virginie

    2015-01-01

    Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a source of nitrogen, thereby improving biomass formation, fermentation efficiency, and cell viability. Thus, the acquisition of FOT genes has favored yeast adaptation to the nitrogen-limited wine fermentation environment. This finding indicates that anthropic environments offer substantial ecological opportunity for evolutionary diversification through gene exchange between distant yeast species. PMID:25750179

  17. Noise minimization in eukaryotic gene expression

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  18. Soil metatranscriptomics for mining eukaryotic heavy metal resistance genes.

    PubMed

    Lehembre, Frédéric; Doillon, Didier; David, Elise; Perrotto, Sandrine; Baude, Jessica; Foulon, Julie; Harfouche, Lamia; Vallon, Laurent; Poulain, Julie; Da Silva, Corinne; Wincker, Patrick; Oger-Desfeux, Christine; Richaud, Pierre; Colpaert, Jan V; Chalot, Michel; Fraissinet-Tachet, Laurence; Blaudez, Damien; Marmeisse, Roland

    2013-10-01

    Heavy metals are pollutants which affect all organisms. Since a small number of eukaryotes have been investigated with respect to metal resistance, we hypothesize that many genes that control this phenomenon remain to be identified. This was tested by screening soil eukaryotic metatranscriptomes which encompass RNA from organisms belonging to the main eukaryotic phyla. Soil-extracted polyadenylated mRNAs were converted into cDNAs and 35 of them were selected for their ability to rescue the metal (Cd or Zn) sensitive phenotype of yeast mutants. Few of the genes belonged to families known to confer metal resistance when overexpressed in yeast. Several of them were homologous to genes that had not been studied in the context of metal resistance. For instance, the BOLA ones, which conferred cross metal (Zn, Co, Cd, Mn) resistance may act by interfering with Fe homeostasis. Other genes, such as those encoding 110- to 130-amino-acid-long, cysteine-rich polypeptides, had no homologues in databases. This study confirms that functional metatranscriptomics represents a powerful approach to address basic biological processes in eukaryotes. The selected genes can be used to probe new pathways involved in metal homeostasis and to manipulate the resistance level of selected organisms.

  19. Protein Subcellular Relocalization Increases the Retention of Eukaryotic Duplicate Genes

    PubMed Central

    Byun, S. Ashley; Singh, Sarabdeep

    2013-01-01

    Gene duplication is widely accepted as a key evolutionary process, leading to new genes and novel protein functions. By providing the raw genetic material necessary for functional expansion, the mechanisms that involve the retention and functional diversification of duplicate genes are one of the central topics in evolutionary and comparative genomics. One proposed source of retention and functional diversification is protein subcellular relocalization (PSR). PSR postulates that changes in the subcellular location of eukaryotic duplicate proteins can positively modify function and therefore be beneficial to the organism. As such, PSR would promote retention of those relocalized duplicates and result in significantly lower death rates compared with death rates of nonrelocalized duplicate pairs. We surveyed both relocalized and nonrelocalized duplicate proteins from the available genomes and proteomes of 59 eukaryotic species and compared their relative death rates over a Ks range between 0 and 1. Using the Cox proportional hazard model, we observed that the death rates of relocalized duplicate pairs were significantly lower than the death rates of the duplicates without relocalization in most eukaryotic species examined in this study. These observations suggest that PSR significantly increases retention of duplicate genes and that it plays an important, but currently underappreciated, role in the evolution of eukaryotic genomes. PMID:24265504

  20. The effect of negative autoregulation on eukaryotic gene expression

    NASA Astrophysics Data System (ADS)

    Nevozhay, Dmitry; Adams, Rhys; Murphy, Kevin; Josic, Kresimir; Balázsi, G. Ábor

    2009-03-01

    Negative autoregulation is a frequent motif in gene regulatory networks, which has been studied extensively in prokaryotes. Nevertheless, some effects of negative feedback on gene expression in eukaryotic transcriptional networks remain unknown. We studied how the strength of negative feedback regulation affects the characteristics of gene expression in yeast cells carrying synthetic transcriptional cascades. We observed a drastic reduction of gene expression noise and a change in the shape of the dose-response curve. We explained these experimentally observed effects by stochastic simulations and a simple set of algebraic equations.

  1. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    PubMed Central

    2011-01-01

    Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts. PMID:21943216

  2. Automatic annotation of eukaryotic genes, pseudogenes and promoters

    PubMed Central

    Solovyev, Victor; Kosarev, Peter; Seledsov, Igor; Vorobyev, Denis

    2006-01-01

    Background The ENCODE gene prediction workshop (EGASP) has been organized to evaluate how well state-of-the-art automatic gene finding methods are able to reproduce the manual and experimental gene annotation of the human genome. We have used Softberry gene finding software to predict genes, pseudogenes and promoters in 44 selected ENCODE sequences representing approximately 1% (30 Mb) of the human genome. Predictions of gene finding programs were evaluated in terms of their ability to reproduce the ENCODE-HAVANA annotation. Results The Fgenesh++ gene prediction pipeline can identify 91% of coding nucleotides with a specificity of 90%. Our automatic pseudogene finder (PSF program) found 90% of the manually annotated pseudogenes and some new ones. The Fprom promoter prediction program identifies 80% of TATA promoters sequences with one false positive prediction per 2,000 base-pairs (bp) and 50% of TATA-less promoters with one false positive prediction per 650 bp. It can be used to identify transcription start sites upstream of annotated coding parts of genes found by gene prediction software. Conclusion We review our software and underlying methods for identifying these three important structural and functional genome components and discuss the accuracy of predictions, recent advances and open problems in annotating genomic sequences. We have demonstrated that our methods can be effectively used for initial automatic annotation of the eukaryotic genome. PMID:16925832

  3. Gene expression in the unicellular eukaryote Trichomonas vaginalis.

    PubMed

    Smith, Alias; Johnson, Patricia

    2011-01-01

    Control of gene expression is essential to the survival of an organism. Here, we review the current state of gene expression research in Trichomonas vaginalis, with particular attention to the progress made since the release of the genome of this unicellular parasite in 2007. The availability of genome data has allowed the study of an array of biological processes, including the role of small nuclear RNAs involved in the splicing of introns, the components of transcriptional complexes and the presence of discrete DNA elements involved in directing transcription. Both evolutionarily conserved and novel features of T. vaginalis serve to inspire further questions aimed at determining the molecular mechanisms used to regulate gene expression in this highly divergent eukaryote. PMID:21511031

  4. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    PubMed

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

  5. Dynamic chromatin: the regulatory domain organization of eukaryotic gene loci.

    PubMed

    Bonifer, C; Hecht, A; Saueressig, H; Winter, D M; Sippel, A E

    1991-10-01

    It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA-sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis-acting regulatory elements. Additionally, we discovered a novel cis-activity of the border regions of the DNAasel sensitive domain (A-elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A-elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A-elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis-acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high

  6. Role of horizontal gene transfer in the evolution of photosynthetic eukaryotes and their plastids.

    PubMed

    Keeling, Patrick J

    2009-01-01

    Plastids are the organelles derived from a cyanobacterium through endosymbiosis. Unlike mitochondria, plastids are not found in all eukaryotes, but their evolution has an added layer of complexity since plastids have moved between eukaryotic lineages by secondary and tertiary endosymbiotic events. This complex history, together with the genetic integration between plastids and their host, has led to many opportunities for gene flow between phylogenetically distinct lineages. Some intracellular transfers do not lead to a protein functioning in a new environment, but many others do and the protein makeup of many plastids appears to have been influenced by exogenous sources as well. Here, different evolutionary sources and cellular destinations of gene flow that has affected the plastid lineage are reviewed. Most horizontal gene transfer (HGT) affecting the modern plastid has taken place via the host nucleus, in the form of genes for plastid-targeted proteins. The impact of this varies greatly from lineage to lineage, but in some cases such transfers can be as high as one fifth of analyzed genes. More rarely, genes have also been transferred to the plastid genome itself, and plastid genes have also been transferred to other non-plant, non-algal lineages. Overall, the proteome of many plastids has emerged as a mosaic of proteins from many sources, some from within the same cell (e.g., cytosolic genes or genes left over from the replacement of an earlier plastid), some from the plastid of other algal lineages, and some from completely unrelated sources. PMID:19271204

  7. Transcriptional activation of heat-shock genes in eukaryotes.

    PubMed

    Tanguay, R M

    1988-06-01

    Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes. In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level. The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes. Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction. This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes. While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE. Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements. However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes. Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner. The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed. Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.

  8. Eukaryotic snoRNAs: a paradigm for gene expression flexibility.

    PubMed

    Dieci, Giorgio; Preti, Milena; Montanini, Barbara

    2009-08-01

    Small nucleolar RNAs (snoRNAs) are one of the most ancient and numerous families of non-protein-coding RNAs (ncRNAs). The main function of snoRNAs - to guide site-specific rRNA modification - is the same in Archaea and all eukaryotic lineages. In contrast, as revealed by recent genomic and RNomic studies, their genomic organization and expression strategies are the most varied. Seemingly snoRNA coding units have adopted, in the course of evolution, all the possible ways of being transcribed, thus providing a unique paradigm of gene expression flexibility. By focusing on representative fungal, plant and animal genomes, we review here all the documented types of snoRNA gene organization and expression, and we provide a comprehensive account of snoRNA expressional freedom by precisely estimating the frequency, in each genome, of each type of genomic organization. We finally discuss the relevance of snoRNA genomic studies for our general understanding of ncRNA family evolution and expression in eukaryotes.

  9. Mimivirus gene promoters exhibit an unprecedented conservation among all eukaryotes.

    PubMed

    Suhre, Karsten; Audic, Stéphane; Claverie, Jean-Michel

    2005-10-11

    The initial analysis of the recently sequenced genome of Acanthamoeba polyphaga Mimivirus, the largest known double-stranded DNA virus, predicted a proteome of size and complexity more akin to small parasitic bacteria than to other nucleocytoplasmic large DNA viruses and identified numerous functions never before described in a virus. It has been proposed that the Mimivirus lineage could have emerged before the individualization of cellular organisms from the three domains of life. An exhaustive in silico analysis of the noncoding moiety of all known viral genomes now uncovers the unprecedented perfect conservation of an AAAATTGA motif in close to 50% of the Mimivirus genes. This motif preferentially occurs in genes transcribed from the predicted leading strand and is associated with functions required early in the viral infectious cycle, such as transcription and protein translation. A comparison with the known promoter of unicellular eukaryotes, amoebal protists in particular, strongly suggests that the AAAATTGA motif is the structural equivalent of the TATA box core promoter element. This element is specific to the Mimivirus lineage and may correspond to an ancestral promoter structure predating the radiation of the eukaryotic kingdoms. This unprecedented conservation of core promoter regions is another exceptional feature of Mimivirus that again raises the question of its evolutionary origin.

  10. A single eubacterial origin of eukaryotic pyruvate: ferredoxin oxidoreductase genes: implications for the evolution of anaerobic eukaryotes.

    PubMed

    Horner, D S; Hirt, R P; Embley, T M

    1999-09-01

    The iron sulfur protein pyruvate: ferredoxin oxidoreductase (PFO) is central to energy metabolism in amitochondriate eukaryotes, including those with hydrogenosomes. Thus, revealing the evolutionary history of PFO is critical to understanding the origin(s) of eukaryote anaerobic energy metabolism. We determined a complete PFO sequence for Spironucleus barkhanus, a large fragment of a PFO sequence from Clostridium pasteurianum, and a fragment of a new PFO from Giardia lamblia. Phylogenetic analyses of eubacterial and eukaryotic PFO genes suggest a complex history for PFO, including possible gene duplications and horizontal transfers among eubacteria. Our analyses favor a common origin for eukaryotic cytosolic and hydrogenosomal PFOs from a single eubacterial source, rather than from separate horizontal transfers as previously suggested. However, with the present sampling of genes and species, we were unable to infer a specific eubacterial sister group for eukaryotic PFO. Thus, we find no direct support for the published hypothesis that the donor of eukaryote PFO was the common alpha-proteobacterial ancestor of mitochondria and hydrogenosomes. We also report that several fungi and protists encode proteins with PFO domains that are likely monophyletic with PFOs from anaerobic protists. In Saccharomyces cerevisiae, PFO domains combine with fragments of other redox proteins to form fusion proteins which participate in methionine biosynthesis. Our results are consistent with the view that PFO, an enzyme previously considered to be specific to energy metabolism in amitochondriate protists, was present in the common ancestor of contemporary eukaryotes and was retained, wholly or in part, during the evolution of oxygen-dependent and mitochondrion-bearing lineages.

  11. Snapshot of the Eukaryotic Gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach

    PubMed Central

    O'Toole, Nicholas; Barboza, Perry S.; Ungerfeld, Emilio; Leigh, Mary Beth; Selinger, L. Brent; Butler, Greg; Tsang, Adrian; McAllister, Tim A.; Forster, Robert J.

    2011-01-01

    Background Herbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches. Methodology/Principal Findings In this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies. Conclusions/Significance The muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes. PMID:21655220

  12. Evolution of the multifaceted eukaryotic akirin gene family

    PubMed Central

    Macqueen, Daniel J; Johnston, Ian A

    2009-01-01

    Background Akirins are nuclear proteins that form part of an innate immune response pathway conserved in Drosophila and mice. This studies aim was to characterise the evolution of akirin gene structure and protein function in the eukaryotes. Results akirin genes are present throughout the metazoa and arose before the separation of animal, plant and fungi lineages. Using comprehensive phylogenetic analysis, coupled with comparisons of conserved synteny and genomic organisation, we show that the intron-exon structure of metazoan akirin genes was established prior to the bilateria and that a single proto-orthologue duplicated in the vertebrates, before the gnathostome-agnathan separation, producing akirin1 and akirin2. Phylogenetic analyses of seven vertebrate gene families with members in chromosomal proximity to both akirin1 and akirin2 were compatible with a common duplication event affecting the genomic neighbourhood of the akirin proto-orthologue. A further duplication of akirins occurred in the teleost lineage and was followed by lineage-specific patterns of paralogue loss. Remarkably, akirins have been independently characterised by five research groups under different aliases and a comparison of the available literature revealed diverse functions, generally in regulating gene expression. For example, akirin was characterised in arthropods as subolesin, an important growth factor and in Drosophila as bhringi, which has an essential myogenic role. In vertebrates, akirin1 was named mighty in mice and was shown to regulate myogenesis, whereas akirin2 was characterised as FBI1 in rats and promoted carcinogenesis, acting as a transcriptional repressor when bound to a 14-3-3 protein. Both vertebrate Akirins have evolved under comparably strict constraints of purifying selection, although a likelihood ratio test predicted that functional divergence has occurred between paralogues. Bayesian and maximum likelihood tests identified amino-acid positions where the rate of

  13. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  14. Whence genes in pieces: reconstruction of the exon-intron gene structures of the last eukaryotic common ancestor and other ancestral eukaryotes.

    PubMed

    Koonin, Eugene V; Csuros, Miklos; Rogozin, Igor B

    2013-01-01

    In eukaryotes, protein-coding sequences are interrupted by non-coding sequences known as introns. During mRNA maturation, introns are excised by the spliceosome and the coding regions, exons, are spliced to form the mature coding region. The intron densities widely differ between eukaryotic lineages, from 6 to 7 introns per kb of coding sequence in vertebrates, some invertebrates and green plants, to only a few introns across the entire genome in many unicellular eukaryotes. Evolutionary reconstructions using maximum likelihood methods suggest intron-rich ancestors for each major group of eukaryotes. For the last common ancestor of animals, the highest intron density of all extant and extinct eukaryotes was inferred, at 120-130% of the human intron density. Furthermore, an intron density within 53-74% of the human values was inferred for the last eukaryotic common ancestor. Accordingly, evolution of eukaryotic genes in all lines of descent involved primarily intron loss, with substantial gain only at the bases of several branches including plants and animals. These conclusions have substantial biological implications indicating that the common ancestor of all modern eukaryotes was a complex organism with a gene architecture resembling those in multicellular organisms. Alternative splicing most likely initially appeared as an inevitable result of splicing errors and only later was employed to generate structural and functional diversification of proteins.

  15. GeneTack database: genes with frameshifts in prokaryotic genomes and eukaryotic mRNA sequences.

    PubMed

    Antonov, Ivan; Baranov, Pavel; Borodovsky, Mark

    2013-01-01

    Database annotations of prokaryotic genomes and eukaryotic mRNA sequences pay relatively low attention to frame transitions that disrupt protein-coding genes. Frame transitions (frameshifts) could be caused by sequencing errors or indel mutations inside protein-coding regions. Other observed frameshifts are related to recoding events (that evolved to control expression of some genes). Earlier, we have developed an algorithm and software program GeneTack for ab initio frameshift finding in intronless genes. Here, we describe a database (freely available at http://topaz.gatech.edu/GeneTack/db.html) containing genes with frameshifts (fs-genes) predicted by GeneTack. The database includes 206 991 fs-genes from 1106 complete prokaryotic genomes and 45 295 frameshifts predicted in mRNA sequences from 100 eukaryotic genomes. The whole set of fs-genes was grouped into clusters based on sequence similarity between fs-proteins (conceptually translated fs-genes), conservation of the frameshift position and frameshift direction (-1, +1). The fs-genes can be retrieved by similarity search to a given query sequence via a web interface, by fs-gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, etc. The largest clusters contain fs-genes with programed frameshifts (related to recoding events).

  16. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes.

    PubMed

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-08-22

    Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes-as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.

  17. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    SciTech Connect

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  18. Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes.

    PubMed

    Ren, Ren; Sun, Yazhou; Zhao, Yue; Geiser, David; Ma, Hong; Zhou, Xiaofan

    2016-09-11

    A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life.

  19. Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes.

    PubMed

    Ren, Ren; Sun, Yazhou; Zhao, Yue; Geiser, David; Ma, Hong; Zhou, Xiaofan

    2016-01-01

    A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life. PMID:27604879

  20. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs.

    PubMed

    Nelles, L; Fang, B L; Volckaert, G; Vandenberghe, A; De Wachter, R

    1984-12-11

    The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.

  1. Gene transfers shaped the evolution of de novo NAD+ biosynthesis in eukaryotes.

    PubMed

    Ternes, Chad M; Schönknecht, Gerald

    2014-09-01

    NAD(+) is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD(+) biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD(+) biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD(+) biosynthesis in eukaryotes was shaped by numerous gene transfers.

  2. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

  3. Eukaryotic origin of a metabolic pathway in virus by horizontal gene transfer.

    PubMed

    Wu, Dong-Dong; Zhang, Ya-Ping

    2011-11-01

    Horizontal gene transfer, the movement of genetic materials across the normal mating barriers between organisms occurs frequently and contributes significantly to the evolution of both eukaryotic and prokaryotic genomes. However, few concurrent transfers of functionally related genes implemented in a pathway from eukaryotes to prokaryotes are observed. Here, we did phylogenetic analyses to support that the genes, i.e. dihydrofolate reductase, glycine hydroxymethyltransferase, and thymidylate synthase involved in thymidylate metabolism, in Hz-1 virus were obtained from insect genome recently by independent horizontal gene transfers. In addition, five other related genes in nucleotide metabolism show evidences of horizontal gene transfers. These genes demonstrate similar expression pattern, and they may have formatted a functionally related pathway (e.g. thymidylate synthesis, and DNA replication) in Hz-1 virus. In conclusion, we provide an example of horizontal gene transfer of functionally related genes in a pathway to prokaryote from eukaryote.

  4. Widespread horizontal gene transfer from double-stranded RNA viruses to eukaryotic nuclear genomes.

    PubMed

    Liu, Huiquan; Fu, Yanping; Jiang, Daohong; Li, Guoqing; Xie, Jiatao; Cheng, Jiasen; Peng, Youliang; Ghabrial, Said A; Yi, Xianhong

    2010-11-01

    Horizontal gene transfer commonly occurs from cells to viruses but rarely occurs from viruses to their host cells, with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genome databases for various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms, including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence of junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely transferred horizontally from viruses to eukaryotic genomes. Furthermore, we present evidence showing that some of the transferred genes are conserved and expressed in eukaryotic organisms and suggesting that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of double-stranded RNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes.

  5. Doxycycline-dependent photoactivated gene expression in eukaryotic systems.

    PubMed

    Cambridge, Sidney B; Geissler, Daniel; Calegari, Federico; Anastassiadis, Konstantinos; Hasan, Mazahir T; Stewart, A Francis; Huttner, Wieland B; Hagen, Volker; Bonhoeffer, Tobias

    2009-07-01

    High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms. PMID:19503080

  6. Adaptive eukaryote-to-eukaryote lateral gene transfer: stress-related genes of algal origin in the closest unicellular relatives of animals.

    PubMed

    Nedelcu, A M; Miles, I H; Fagir, A M; Karol, K

    2008-11-01

    In addition to mutation, gene duplication and recombination, the transfer of genetic material between unrelated species is now regarded as a potentially significant player in the shaping of extant genomes and the evolution and diversification of life. Although this is probably true for prokaryotes, the extent of such genetic exchanges in eukaryotes (especially eukaryote-to-eukaryote transfers) is more controversial and the selective advantage and evolutionary impact of such events are less documented. A laterally transferred gene could either be added to the gene complement of the recipient or replace the recipient's homologue; whereas gene replacements can be either adaptive or stochastic, gene additions are most likely adaptive. Here, we report the finding of four stress-related genes (two ascorbate peroxidase and two metacaspase genes) of algal origin in the closest unicellular relatives of animals, the choanoflagellates. At least three of these sequences represent additions to the choanoflagellate gene complement, which is consistent with these transfers being adaptive. We suggest that these laterally acquired sequences could have provided the primitive choanoflagellates with additional or more efficient means to cope with stress, especially in relation to adapting to freshwater environments and/or sessile or colonial lifestyles.

  7. Adaptive eukaryote-to-eukaryote lateral gene transfer: stress-related genes of algal origin in the closest unicellular relatives of animals.

    PubMed

    Nedelcu, A M; Miles, I H; Fagir, A M; Karol, K

    2008-11-01

    In addition to mutation, gene duplication and recombination, the transfer of genetic material between unrelated species is now regarded as a potentially significant player in the shaping of extant genomes and the evolution and diversification of life. Although this is probably true for prokaryotes, the extent of such genetic exchanges in eukaryotes (especially eukaryote-to-eukaryote transfers) is more controversial and the selective advantage and evolutionary impact of such events are less documented. A laterally transferred gene could either be added to the gene complement of the recipient or replace the recipient's homologue; whereas gene replacements can be either adaptive or stochastic, gene additions are most likely adaptive. Here, we report the finding of four stress-related genes (two ascorbate peroxidase and two metacaspase genes) of algal origin in the closest unicellular relatives of animals, the choanoflagellates. At least three of these sequences represent additions to the choanoflagellate gene complement, which is consistent with these transfers being adaptive. We suggest that these laterally acquired sequences could have provided the primitive choanoflagellates with additional or more efficient means to cope with stress, especially in relation to adapting to freshwater environments and/or sessile or colonial lifestyles. PMID:18717747

  8. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    PubMed

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

  9. Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

    PubMed Central

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M.; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies. PMID:24516555

  10. Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes.

    PubMed

    Archibald, J M; Logsdon, J M; Doolittle, W F

    2000-10-01

    Chaperonins are oligomeric protein-folding complexes which are divided into two distantly related structural classes. Group I chaperonins (called GroEL/cpn60/hsp60) are found in bacteria and eukaryotic organelles, while group II chaperonins are present in archaea and the cytoplasm of eukaryotes (called CCT/TriC). While archaea possess one to three chaperonin subunit-encoding genes, eight distinct CCT gene families (paralogs) have been characterized in eukaryotes. We are interested in determining when during eukaryotic evolution the multiple gene duplications producing the CCT subunits occurred. We describe the sequence and phylogenetic analysis of five CCT genes from TRICHOMONAS: vaginalis and seven from GIARDIA: lamblia, representatives of amitochondriate protist lineages thought to have diverged early from other eukaryotes. Our data show that the gene duplications producing the eight CCT paralogs took place prior to the organismal divergence of TRICHOMONAS: and GIARDIA: from other eukaryotes. Thus, these divergent protists likely possess completely hetero-oligomeric CCT complexes like those in yeast and mammalian cells. No close phylogenetic relationship between the archaeal chaperonins and specific CCT subunits was observed, suggesting that none of the CCT gene duplications predate the divergence of archaea and eukaryotes. The duplications producing the CCTdelta and CCTepsilon subunits, as well as CCTalpha, CCTbeta, and CCTeta, are the most recent in the CCT gene family. Our analyses show significant differences in the rates of evolution of archaeal chaperonins compared with the eukaryotic CCTs, as well as among the different CCT subunits themselves. We discuss these results in light of current views on the origin, evolution, and function of CCT complexes.

  11. A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples.

    PubMed

    Dollive, Serena; Peterfreund, Gregory L; Sherrill-Mix, Scott; Bittinger, Kyle; Sinha, Rohini; Hoffmann, Christian; Nabel, Christopher S; Hill, David A; Artis, David; Bachman, Michael A; Custers-Allen, Rebecca; Grunberg, Stephanie; Wu, Gary D; Lewis, James D; Bushman, Frederic D

    2012-07-03

    Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

  12. Translational Control of Viral Gene Expression in Eukaryotes

    PubMed Central

    Gale, Michael; Tan, Seng-Lai; Katze, Michael G.

    2000-01-01

    As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell. PMID:10839817

  13. Evolutionary diversity of eukaryotic small-subunit rRNA genes.

    PubMed

    Sogin, M L; Elwood, H J; Gunderson, J H

    1986-03-01

    The small-subunit rRNA gene sequences of the flagellated protists Euglena gracilis and Trypanosoma brucei were determined and compared to those of other eukaryotes. A phylogenetic tree was constructed in which the earliest branching among the eukaryotes is represented by E. gracilis. The E. gracilis divergence far antedates a period of massive evolutionary radiation that gave rise to the plants, animals, fungi, and certain groups of protists such as ciliates and the acanthamoebae. The genetic diversity in this collection of eukaryotes is seen to exceed that displayed within either the eubacterial or the archaebacterial lines of descent.

  14. Lateral gene transfers and the origins of the eukaryote proteome: a view from microbial parasites

    PubMed Central

    Hirt, Robert P; Alsmark, Cecilia; Embley, T Martin

    2015-01-01

    Our knowledge of the extent and functional impact of lateral gene transfer (LGT) from prokaryotes to eukaryotes, outside of endosymbiosis, is still rather limited. Here we review the recent literature, focusing mainly on microbial parasites, indicating that LGT from diverse prokaryotes has played a significant role in the evolution of a number of lineages, and by extension throughout eukaryotic evolution. As might be expected, taxonomic biases for donor prokaryotes indicate that shared habitat is a major factor driving transfers. The LGTs identified predominantly affect enzymes from metabolic pathways, but over a third of LGT are genes for putative proteins of unknown function. Finally, we discuss the difficulties in analysing LGT among eukaryotes and suggest that high-throughput methodologies integrating different approaches are needed to achieve a more global understanding of the importance of LGT in eukaryotic evolution. PMID:25483352

  15. Endosymbiotic and horizontal gene transfer in microbial eukaryotes: Impacts on cell evolution and the tree of life.

    PubMed

    Chan, Cheong Xin; Bhattacharya, Debashish; Reyes-Prieto, Adrian

    2012-03-01

    The evolution of microbial eukaryotes, in particular of photosynthetic lineages, is complicated by multiple instances of endosymbiotic and horizontal gene transfer (E/HGT) resulting from plastid origin(s). Our recent analysis of diatom membrane transporters provides evidence of red and/or green algal origins of 172 of the genes encoding these proteins (ca. 25% of the examined phylogenies), with the majority putatively derived from green algae. These data suggest that E/HGT has been an important driver of evolutionary innovation among diatoms (and likely other stramenopiles), and lend further support to the hypothesis of an ancient, cryptic green algal endosymbiosis in "chromalveolate" lineages. Here, we discuss the implications of our findings on the understanding of eukaryote evolution and inference of the tree of life.

  16. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  17. A cyanobacterial gene in nonphotosynthetic protists--an early chloroplast acquisition in eukaryotes?

    PubMed

    Andersson, Jan O; Roger, Andrew J

    2002-01-22

    Since the incorporation of mitochondria and chloroplasts (plastids) into the eukaryotic cell by endosymbiosis, genes have been transferred from the organellar genomes to the nucleus of the host, via an ongoing process known as endosymbiotic gene transfer. Accordingly, in photosynthetic eukaryotes, nuclear genes with cyanobacterial affinity are believed to have originated from endosymbiotic gene transfer from chloroplasts. Analysis of the Arabidopsis thaliana genome has shown that a significant fraction (2%-9%) of the nuclear genes have such an endosymbiotic origin. Recently, it was argued that 6-phosphogluconate dehydrogenase (gnd)-the second enzyme in the oxidative pentose phosphate pathway-was one such example. Here we show that gnd genes with cyanobacterial affinity also are present in several nonphotosynthetic protistan lineages, such as Heterolobosea, Apicomplexa, and parasitic Heterokonta. Current data cannot definitively resolve whether these groups acquired the gnd gene by primary and/or secondary endosymbiosis or via an independent lateral gene transfer event. Nevertheless, our data suggest that chloroplasts were introduced into eukaryotes much earlier than previously thought and that several major groups of heterotrophic eukaryotes have secondarily lost photosynthetic plastids.

  18. Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

    PubMed Central

    Villarreal, L P

    1991-01-01

    The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and

  19. Comparative genomics of protists: new insights into the evolution of eukaryotic signal transduction and gene regulation.

    PubMed

    Anantharaman, Vivek; Iyer, Lakshminarayan M; Aravind, L

    2007-01-01

    Data from protist genomes suggest that eukaryotes show enormous variability in their gene complements, especially of genes coding regulatory proteins. Overall counts of eukaryotic signaling proteins show weak nonlinear scaling with proteome size, but individual superfamilies of signaling domains might show vast expansions in certain protists. Alteration of domain architectural complexity of signaling proteins and repeated lineage-specific reshaping of architectures might have played a major role in the emergence of new signaling interactions in different eukaryotes. Lateral transfer of various signaling domains from bacteria or from hosts, in parasites such as apicomplexans, appears to also have played a major role in the origin of new functional networks. Lineage-specific expansion of regulatory proteins, particularly of transcription factors, has played a critical role in the adaptive radiation of different protist lineages. Comparative genomics allows objective reconstruction of the ancestral conditions and subsequent diversification of several regulatory systems involved in phosphorylation, cyclic nucleotide signaling, Ubiquitin conjugation, chromatin remodeling, and posttranscriptional gene silencing.

  20. Common DNA structural features exhibited by eukaryotic ribosomal gene promoters.

    PubMed Central

    Marilley, M; Pasero, P

    1996-01-01

    Nucleotide sequences of DNA regions containing eukaryotic ribosomal promoters were analysed using strategies designed to reveal sequence-directed structural features. DNA curvature, duplex stability and pattern of twist angle variation were studied by computer modelling. Although ribosomal promoters are known to lack sequence homology (unless very closely related species are considered), investigation of these structural characteristics uncovered striking homologies in all the taxonomic groups examined so far. This wide conservation of DNA structures, while DNA sequence is not conserved, suggests that the determined structures are fundamental for ribosomal promoter function. Moreover, this result agrees well with the recent observations showing that RNA polymerase I transcription factors have not evolved as intensively as previously suspected. PMID:8710487

  1. Analysis of evolution of exon-intron structure of eukaryotic genes.

    PubMed

    Rogozin, Igor B; Sverdlov, Alexander V; Babenko, Vladimir N; Koonin, Eugene V

    2005-06-01

    The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were

  2. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

    SciTech Connect

    Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B

    2011-08-08

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  3. Three Dimensional Organization of Genome Might Have Guided the Dynamics of Gene Order Evolution in Eukaryotes

    PubMed Central

    Bagadia, Meenakshi; Singh, Arashdeep; Singh Sandhu, Kuljeet

    2016-01-01

    In eukaryotes, genes are nonrandomly organized into short gene-dense regions or “gene-clusters” interspersed by long gene-poor regions. How these gene-clusters have evolved is not entirely clear. Gene duplication may not account for all the gene-clusters since the genes in most of the clusters do not exhibit significant sequence similarity. In this study, using genome-wide data sets from budding yeast, fruit-fly, and human, we show that: 1) long-range evolutionary repositioning of genes strongly associate with their spatial proximity in the nucleus; 2) presence of evolutionary DNA break-points at involved loci hints at their susceptibility to undergo long-range genomic rearrangements; and 3) correlated epigenetic and transcriptional states of engaged genes highlight the underlying evolutionary constraints. The significance of observation 1, 2, and 3 are particularly stronger for the instances of inferred evolutionary gain, as compared with loss, of linear gene-clustering. These observations suggest that the long-range genomic rearrangements guided through 3D genome organization might have contributed to the evolution of gene order. We further hypothesize that the evolution of linear gene-clusters in eukaryotic genomes might have been mediated through spatial interactions among distant loci in order to optimize co-ordinated regulation of genes. We model this hypothesis through a heuristic model of gene-order evolution. PMID:26957031

  4. Biosurfactant gene clusters in eukaryotes: regulation and biotechnological potential.

    PubMed

    Roelants, Sophie L K W; De Maeseneire, Sofie L; Ciesielska, Katarzyna; Van Bogaert, Inge N A; Soetaert, Wim

    2014-04-01

    Biosurfactants (BSs) are a class of secondary metabolites representing a wide variety of structures that can be produced from renewable feedstock by a wide variety of micro-organisms. They have (potential) applications in the medical world, personal care sector, mining processes, food industry, cosmetics, crop protection, pharmaceuticals, bio-remediation, household detergents, paper and pulp industry, textiles, paint industries, etc. Especially glycolipid BSs like sophorolipids (SLs), rhamnolipids (RLs), mannosylerythritol lipids (MELs) and cellobioselipids (CBLs) have been described to provide significant opportunities to (partially) replace chemical surfactants. The major two factors currently limiting the penetration of BSs into the market are firstly the limited structural variety and secondly the rather high production price linked with the productivity. One of the keys to resolve the above mentioned bottlenecks can be found in the genetic engineering of natural producers. This could not only result in more efficient (economical) recombinant producers, but also in a diversification of the spectrum of available BSs as such resolving both limiting factors at once. Unraveling the genetics behind the biosynthesis of these interesting biological compounds is indispensable for the tinkering, fine tuning and rearrangement of these biological pathways with the aim of obtaining higher yields and a more extensive structural variety. Therefore, this review focuses on recent developments in the investigation of the biosynthesis, genetics and regulation of some important members of the family of the eukaryotic glycolipid BSs (MELs, CBLs and SLs). Moreover, recent biotechnological achievements and the industrial potential of engineered strains are discussed.

  5. Biosurfactant gene clusters in eukaryotes: regulation and biotechnological potential.

    PubMed

    Roelants, Sophie L K W; De Maeseneire, Sofie L; Ciesielska, Katarzyna; Van Bogaert, Inge N A; Soetaert, Wim

    2014-04-01

    Biosurfactants (BSs) are a class of secondary metabolites representing a wide variety of structures that can be produced from renewable feedstock by a wide variety of micro-organisms. They have (potential) applications in the medical world, personal care sector, mining processes, food industry, cosmetics, crop protection, pharmaceuticals, bio-remediation, household detergents, paper and pulp industry, textiles, paint industries, etc. Especially glycolipid BSs like sophorolipids (SLs), rhamnolipids (RLs), mannosylerythritol lipids (MELs) and cellobioselipids (CBLs) have been described to provide significant opportunities to (partially) replace chemical surfactants. The major two factors currently limiting the penetration of BSs into the market are firstly the limited structural variety and secondly the rather high production price linked with the productivity. One of the keys to resolve the above mentioned bottlenecks can be found in the genetic engineering of natural producers. This could not only result in more efficient (economical) recombinant producers, but also in a diversification of the spectrum of available BSs as such resolving both limiting factors at once. Unraveling the genetics behind the biosynthesis of these interesting biological compounds is indispensable for the tinkering, fine tuning and rearrangement of these biological pathways with the aim of obtaining higher yields and a more extensive structural variety. Therefore, this review focuses on recent developments in the investigation of the biosynthesis, genetics and regulation of some important members of the family of the eukaryotic glycolipid BSs (MELs, CBLs and SLs). Moreover, recent biotechnological achievements and the industrial potential of engineered strains are discussed. PMID:24531239

  6. Evolutionary analyses of the small subunit of glutamate synthase: gene order conservation, gene fusions, and prokaryote-to-eukaryote lateral gene transfers.

    PubMed

    Andersson, Jan O; Roger, Andrew J

    2002-04-01

    Lateral gene transfer has been identified as an important mode of genome evolution within prokaryotes. Except for the special case of gene transfer from organelle genomes to the eukaryotic nucleus, only a few cases of lateral gene transfer involving eukaryotes have been described. Here we present phylogenetic and gene order analyses on the small subunit of glutamate synthase (encoded by gltD) and its homologues, including the large subunit of sulfide dehydrogenase (encoded by sudA). The scattered distribution of the sudA and sudB gene pair and the phylogenetic analysis strongly suggest that lateral gene transfer was involved in the propagation of the genes in the three domains of life. One of these transfers most likely occurred between a prokaryote and an ancestor of diplomonad protists. Furthermore, phylogenetic analyses indicate that the gene for the small subunit of glutamate synthase was transferred from a low-GC gram-positive bacterium to a common ancestor of animals, fungi, and plants. Interestingly, in both examples, the eukaryotes encode a single gene that corresponds to a conserved operon structure in prokaryotes. Our analyses, together with several recent publications, show that lateral gene transfers from prokaryotes to unicellular eukaryotes occur with appreciable frequency. In the case of the genes for sulfide dehydrogenase, the transfer affected only a limited group of eukaryotes--the diplomonads--while the transfer of the glutamate synthase gene probably happened earlier in evolution and affected a wider range of eukaryotes.

  7. Gene flow and biological conflict systems in the origin and evolution of eukaryotes.

    PubMed

    Aravind, L; Anantharaman, Vivek; Zhang, Dapeng; de Souza, Robson F; Iyer, Lakshminarayan M

    2012-01-01

    The endosymbiotic origin of eukaryotes brought together two disparate genomes in the cell. Additionally, eukaryotic natural history has included other endosymbiotic events, phagotrophic consumption of organisms, and intimate interactions with viruses and endoparasites. These phenomena facilitated large-scale lateral gene transfer and biological conflicts. We synthesize information from nearly two decades of genomics to illustrate how the interplay between lateral gene transfer and biological conflicts has impacted the emergence of new adaptations in eukaryotes. Using apicomplexans as example, we illustrate how lateral transfer from animals has contributed to unique parasite-host interfaces comprised of adhesion- and O-linked glycosylation-related domains. Adaptations, emerging due to intense selection for diversity in the molecular participants in organismal and genomic conflicts, being dispersed by lateral transfer, were subsequently exapted for eukaryote-specific innovations. We illustrate this using examples relating to eukaryotic chromatin, RNAi and RNA-processing systems, signaling pathways, apoptosis and immunity. We highlight the major contributions from catalytic domains of bacterial toxin systems to the origin of signaling enzymes (e.g., ADP-ribosylation and small molecule messenger synthesis), mutagenic enzymes for immune receptor diversification and RNA-processing. Similarly, we discuss contributions of bacterial antibiotic/siderophore synthesis systems and intra-genomic and intra-cellular selfish elements (e.g., restriction-modification, mobile elements and lysogenic phages) in the emergence of chromatin remodeling/modifying enzymes and RNA-based regulation. We develop the concept that biological conflict systems served as evolutionary "nurseries" for innovations in the protein world, which were delivered to eukaryotes via lateral gene flow to spur key evolutionary innovations all the way from nucleogenesis to lineage-specific adaptations. PMID:22919680

  8. Eukaryotic mRNA decay: methodologies, pathways, and links to other stages of gene expression.

    PubMed

    Pérez-Ortín, José E; Alepuz, Paula; Chávez, Sebastián; Choder, Mordechai

    2013-10-23

    mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.

  9. Gene targeting for chromosome engineering applications in eukaryotic cells.

    PubMed

    Lyznik, Leszek A; Dress, Virginia

    2008-01-01

    As biotechnology advances, there is an increasing need to develop new technologies that may assist in more precise genetic engineering manipulations. Whether a placement of single genes in the proper chromosomal context, stacking a number of genes in the same chromosomal locus, rearrangement of existing chromosomal elements, or a global reconfiguration of the chromosomal structures is contemplated, the new genetic tools being developed provide technical capabilities to achieve goals that were only theoretical not long ago. We use examples of recent patent literature (issued patents and published patent applications) to illustrate trends in this fast advancing area of genetic technology. If one wants to engage in the development and utilization of such technologies, the complexity of genetic manipulations requires a careful evaluation and navigation across the legal/patent landscape of chromosomal modification/remodeling. While this review is mostly focused on the basic laboratory tools of chromosomal manipulations, their specific applications for biomedical, pharmaceutical, or agricultural purposes may deserve an additional compilation.

  10. Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae.

    PubMed

    O'Neill, E C; Saalbach, G; Field, R A

    2016-01-01

    Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered. PMID:27480684

  11. An overabundance of phase 0 introns immediately after the start codon in eukaryotic genes

    PubMed Central

    Nielsen, Henrik; Wernersson, Rasmus

    2006-01-01

    Background A knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. Despite this, there has been relatively little focus on the distribution of intron positions in genes. Results In proteins with signal peptides, there is an overabundance of phase 1 introns around the region of the signal peptide cleavage site. This has been described before. But in proteins without signal peptides, a novel phenomenon is observed: There is a sharp peak of phase 0 intron positions immediately following the start codon, i.e. between codons 1 and 2. This effect is seen in a wide range of eukaryotes: Vertebrates, arthropods, fungi, and flowering plants. Proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. In addition, there is a peak of phase 0 introns at position 5 in Drosophila genes with signal peptides, predominantly representing cuticle proteins. Conclusion There is an overabundance of phase 0 introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. We give a detailed description of these start codon introns and the proteins that contain them. PMID:17034638

  12. A study of eukaryotic response mechanisms to atmospheric pressure cold plasma by using Saccharomyces cerevisiae single gene mutants

    SciTech Connect

    Feng Hongqing; Wang Ruixue; Sun Peng; Wu Haiyan; Liu Qi; Li Fangting; Fang Jing; Zhang Jue; Zhu Weidong

    2010-09-27

    The mechanisms of eukaryotic cell response to cold plasma are studied. A series of single gene mutants of eukaryotic model organism Saccharomyces cerevisiae are used to compare their sensitivity to plasma treatment with the wild type. We examined 12 mutants in the oxidative stress pathway and the cell cycle pathway, in which 8 are found to be hypersensitive to plasma processing. The mutated genes' roles in the two pathways are analyzed to understand the biological response mechanisms of plasma treatment. The results demonstrate that genes from both pathways are needed for the eukaryotic cells to survive the complex plasma treatment.

  13. WebAUGUSTUS—a web service for training AUGUSTUS and predicting genes in eukaryotes

    PubMed Central

    Hoff, Katharina J.; Stanke, Mario

    2013-01-01

    The prediction of protein coding genes is an important step in the annotation of newly sequenced and assembled genomes. AUGUSTUS is one of the most accurate tools for eukaryotic gene prediction. Here, we present WebAUGUSTUS, a web interface for training AUGUSTUS and predicting genes with AUGUSTUS. Depending on the needs of the user, WebAUGUSTUS generates training gene structures automatically. Besides a genome file, either a file with expressed sequence tags or a file with protein sequences is required for this step. Alternatively, it is possible to submit an externally generated training gene structure file and a genome file. The web service optimizes AUGUSTUS parameters and predicts genes with those parameters. WebAUGUSTUS is available at http://bioinf.uni-greifswald.de/webaugustus. PMID:23700307

  14. Differential gene expression in Giardia lamblia under oxidative stress: significance in eukaryotic evolution.

    PubMed

    Raj, Dibyendu; Ghosh, Esha; Mukherjee, Avik K; Nozaki, Tomoyoshi; Ganguly, Sandipan

    2014-02-10

    Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production. To study differential gene expression under three types of oxidative stress, a Giardia genomic DNA array was constructed and hybridized with labeled cDNA of cells with or without stress. The transcriptomic data has been analyzed and further validated using real time PCR. We identified that out of 9216 genes represented on the array, more than 200 genes encoded proteins with functions in metabolism, oxidative stress management, signaling, reproduction and cell division, programmed cell death and cytoskeleton. We recognized genes modulated by at least ≥ 2 fold at a significant time point in response to oxidative stress. The study has highlighted the genes that are differentially expressed during the three experimental conditions which regulate the stress management pathway differently to achieve redox homeostasis. Identification of some unique genes in oxidative stress regulation may help in new drug designing for this common enteric parasite prone to

  15. Microsatellites in the Eukaryotic DNA Mismatch Repair Genes as Modulators of Evolutionary Mutation Rate

    NASA Technical Reports Server (NTRS)

    Chang, Dong Kyung; Metzgar, David; Wills, Christopher; Boland, C. Richard

    2003-01-01

    All "minor" components of the human DNA mismatch repair (MMR) system-MSH3, MSH6, PMS2, and the recently discovered MLH3-contain mononucleotide microsatellites in their coding sequences. This intriguing finding contrasts with the situation found in the major components of the DNA MMR system-MSH2 and MLH1-and, in fact, most human genes. Although eukaryotic genomes are rich in microsatellites, non-triplet microsatellites are rare in coding regions. The recurring presence of exonal mononucleotide repeat sequences within a single family of human genes would therefore be considered exceptional.

  16. Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist.

    PubMed

    Katz, Laura A

    2015-09-26

    While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence-absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages.

  17. Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist

    PubMed Central

    Katz, Laura A.

    2015-01-01

    While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages. PMID:26323756

  18. ANALYSIS OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GENES REVEALS THE COMPLEX EVOLUTIONARY HISTORY OF A MICROBIAL EUKARYOTE().

    PubMed

    Chan, Cheong Xin; Soares, Marcelo B; Bonaldo, Maria F; Wisecaver, Jennifer H; Hackett, Jeremiah D; Anderson, Donald M; Erdner, Deana L; Bhattacharya, Debashish

    2012-10-01

    Microbial eukaryotes may extinguish much of their nuclear phylogenetic history due to endosymbiotic/horizontal gene transfer (E/HGT). We studied E/HGT in 32,110 contigs of expressed sequence tags (ESTs) from the dinoflagellate Alexandrium tamarense (Dinophyceae) using a conservative phylogenomic approach. The vast majority of predicted proteins (86.4%) in this alga are novel or dinoflagellate-specific. We searched for putative homologs of these predicted proteins against a taxonomically broadly sampled protein database that includes all currently available data from algae and protists and reconstructed a phylogeny from each of the putative homologous protein sets. Of the 2,523 resulting phylogenies, 14-17% are potentially impacted by E/HGT involving both prokaryote and eukaryote lineages, with 2-4% showing clear evidence of reticulate evolution. The complex evolutionary histories of the remaining proteins, many of which may also have been affected by E/HGT, cannot be interpreted using our approach with currently available gene data. We present empirical evidence of reticulate genome evolution that combined with inadequate or highly complex phylogenetic signal in many proteins may impede genome-wide approaches to infer the tree of microbial eukaryotes.

  19. A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis.

    PubMed

    Ramesh, Marilee A; Malik, Shehre-Banoo; Logsdon, John M

    2005-01-26

    Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.

  20. Multiple occurrences of giant virus core genes acquired by eukaryotic genomes: the visible part of the iceberg?

    PubMed

    Filée, Jonathan

    2014-10-01

    Giant Viruses are a widespread group of viruses, characterized by huge genomes composed of a small subset of ancestral, vertically inherited core genes along with a large body of highly variable genes. In this study, I report the acquisition of 23 core ancestral Giant Virus genes by diverse eukaryotic species including various protists, a moss and a cnidarian. The viral genes are inserted in large scaffolds or chromosomes with intron-rich, eukaryotic-like genomic contexts, refuting the possibility of DNA contaminations. Some of these genes are expressed and in the cryptophyte alga Guillardia theta, a possible non-homologous displacement of the eukaryotic DNA primase by a viral D5 helicase/primase is documented. As core Giant Virus genes represent only a tiny fraction of the total genomic repertoire of these viruses, these results suggest that Giant Viruses represent an underestimated source of new genes and functions for their hosts.

  1. Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity.

    PubMed

    Monier, Adam; Sudek, Sebastian; Fast, Naomi M; Worden, Alexandra Z

    2013-09-01

    Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes.

  2. Polycistronic peptide coding genes in eukaryotes--how widespread are they?

    PubMed

    Tautz, Diethard

    2009-01-01

    The classical textbook assumption for the structure of an eukaryotic gene is that it codes for a single polypeptide of more than 100 amino acids in length. This is also the implicit assumption in most gene annotation pipelines. A gene family has now been discovered in insects that shows that an eukaryotic mRNA can code for peptides as short as eleven amino acids and that a single mRNA can code for several such peptides. This raises the question whether short open reading frames might also have a functional potential in other mRNAs, in particular those that occur in the 5'-UTR of many mRNAs. A number of these have been shown to act in cis to regulate the translation of the main open reading frame of the mRNA. But there may be others that could act in trans on other biological processes. The question of how many peptide-coding genes may exist is therefore worth revisiting. This poses new bioinformatic challenges that can only be resolved through multiple genome comparisons within a range of evolutionary distances. PMID:19074495

  3. Identification of minimal eukaryotic introns through GeneBase, a user-friendly tool for parsing the NCBI Gene databank.

    PubMed

    Piovesan, Allison; Caracausi, Maria; Ricci, Marco; Strippoli, Pierluigi; Vitale, Lorenza; Pelleri, Maria Chiara

    2015-12-01

    We have developed GeneBase, a full parser of the National Center for Biotechnology Information (NCBI) Gene database, which generates a fully structured local database with an intuitive user-friendly graphic interface for personal computers. Features of all the annotated eukaryotic genes are accessible through three main software tables, including for each entry details such as the gene summary, the gene exon/intron structure and the specific Gene Ontology attributions. The structuring of the data, the creation of additional calculation fields and the integration with nucleotide sequences allow users to make many types of comparisons and calculations that are useful for data retrieval and analysis. We provide an original example analysis of the existing introns across all the available species, through which the classic biological problem of the 'minimal intron' may find a solution using available data. Based on all currently available data, we can define the shortest known eukaryotic GT-AG intron length, setting the physical limit at the 30 base pair intron belonging to the human MST1L gene. This 'model intron' will shed light on the minimal requirement elements of recognition used for conventional splicing functioning. Remarkably, this size is indeed consistent with the sum of the splicing consensus sequence lengths.

  4. Identification of minimal eukaryotic introns through GeneBase, a user-friendly tool for parsing the NCBI Gene databank

    PubMed Central

    Piovesan, Allison; Caracausi, Maria; Ricci, Marco; Strippoli, Pierluigi; Vitale, Lorenza; Pelleri, Maria Chiara

    2015-01-01

    We have developed GeneBase, a full parser of the National Center for Biotechnology Information (NCBI) Gene database, which generates a fully structured local database with an intuitive user-friendly graphic interface for personal computers. Features of all the annotated eukaryotic genes are accessible through three main software tables, including for each entry details such as the gene summary, the gene exon/intron structure and the specific Gene Ontology attributions. The structuring of the data, the creation of additional calculation fields and the integration with nucleotide sequences allow users to make many types of comparisons and calculations that are useful for data retrieval and analysis. We provide an original example analysis of the existing introns across all the available species, through which the classic biological problem of the ‘minimal intron’ may find a solution using available data. Based on all currently available data, we can define the shortest known eukaryotic GT-AG intron length, setting the physical limit at the 30 base pair intron belonging to the human MST1L gene. This ‘model intron’ will shed light on the minimal requirement elements of recognition used for conventional splicing functioning. Remarkably, this size is indeed consistent with the sum of the splicing consensus sequence lengths. PMID:26581719

  5. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

    PubMed

    Zielinski, Brian L; Allen, Andrew E; Carpenter, Edward J; Coles, Victoria J; Crump, Byron C; Doherty, Mary; Foster, Rachel A; Goes, Joaquim I; Gomes, Helga R; Hood, Raleigh R; McCrow, John P; Montoya, Joseph P; Moustafa, Ahmed; Satinsky, Brandon M; Sharma, Shalabh; Smith, Christa B; Yager, Patricia L; Paul, John H

    2016-01-01

    The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as

  6. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

    PubMed

    Zielinski, Brian L; Allen, Andrew E; Carpenter, Edward J; Coles, Victoria J; Crump, Byron C; Doherty, Mary; Foster, Rachel A; Goes, Joaquim I; Gomes, Helga R; Hood, Raleigh R; McCrow, John P; Montoya, Joseph P; Moustafa, Ahmed; Satinsky, Brandon M; Sharma, Shalabh; Smith, Christa B; Yager, Patricia L; Paul, John H

    2016-01-01

    The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as

  7. Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume

    PubMed Central

    Allen, Andrew E.; Carpenter, Edward J.; Coles, Victoria J.; Crump, Byron C.; Doherty, Mary; Foster, Rachel A.; Goes, Joaquim I.; Gomes, Helga R.; Hood, Raleigh R.; McCrow, John P.; Montoya, Joseph P.; Moustafa, Ahmed; Satinsky, Brandon M.; Sharma, Shalabh; Smith, Christa B.; Yager, Patricia L.; Paul, John H.

    2016-01-01

    The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 μm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as

  8. Origin of eukaryotes from within archaea, archaeal eukaryome and bursts of gene gain: eukaryogenesis just made easier?

    PubMed

    Koonin, Eugene V

    2015-09-26

    The origin of eukaryotes is a fundamental, forbidding evolutionary puzzle. Comparative genomic analysis clearly shows that the last eukaryotic common ancestor (LECA) possessed most of the signature complex features of modern eukaryotic cells, in particular the mitochondria, the endomembrane system including the nucleus, an advanced cytoskeleton and the ubiquitin network. Numerous duplications of ancestral genes, e.g. DNA polymerases, RNA polymerases and proteasome subunits, also can be traced back to the LECA. Thus, the LECA was not a primitive organism and its emergence must have resulted from extensive evolution towards cellular complexity. However, the scenario of eukaryogenesis, and in particular the relationship between endosymbiosis and the origin of eukaryotes, is far from being clear. Four recent developments provide new clues to the likely routes of eukaryogenesis. First, evolutionary reconstructions suggest complex ancestors for most of the major groups of archaea, with the subsequent evolution dominated by gene loss. Second, homologues of signature eukaryotic proteins, such as actin and tubulin that form the core of the cytoskeleton or the ubiquitin system, have been detected in diverse archaea. The discovery of this 'dispersed eukaryome' implies that the archaeal ancestor of eukaryotes was a complex cell that might have been capable of a primitive form of phagocytosis and thus conducive to endosymbiont capture. Third, phylogenomic analyses converge on the origin of most eukaryotic genes of archaeal descent from within the archaeal evolutionary tree, specifically, the TACK superphylum. Fourth, evidence has been presented that the origin of the major archaeal phyla involved massive acquisition of bacterial genes. Taken together, these findings make the symbiogenetic scenario for the origin of eukaryotes considerably more plausible and the origin of the organizational complexity of eukaryotic cells more readily explainable than they appeared until recently.

  9. Origin of eukaryotes from within archaea, archaeal eukaryome and bursts of gene gain: eukaryogenesis just made easier?

    PubMed Central

    Koonin, Eugene V.

    2015-01-01

    The origin of eukaryotes is a fundamental, forbidding evolutionary puzzle. Comparative genomic analysis clearly shows that the last eukaryotic common ancestor (LECA) possessed most of the signature complex features of modern eukaryotic cells, in particular the mitochondria, the endomembrane system including the nucleus, an advanced cytoskeleton and the ubiquitin network. Numerous duplications of ancestral genes, e.g. DNA polymerases, RNA polymerases and proteasome subunits, also can be traced back to the LECA. Thus, the LECA was not a primitive organism and its emergence must have resulted from extensive evolution towards cellular complexity. However, the scenario of eukaryogenesis, and in particular the relationship between endosymbiosis and the origin of eukaryotes, is far from being clear. Four recent developments provide new clues to the likely routes of eukaryogenesis. First, evolutionary reconstructions suggest complex ancestors for most of the major groups of archaea, with the subsequent evolution dominated by gene loss. Second, homologues of signature eukaryotic proteins, such as actin and tubulin that form the core of the cytoskeleton or the ubiquitin system, have been detected in diverse archaea. The discovery of this ‘dispersed eukaryome’ implies that the archaeal ancestor of eukaryotes was a complex cell that might have been capable of a primitive form of phagocytosis and thus conducive to endosymbiont capture. Third, phylogenomic analyses converge on the origin of most eukaryotic genes of archaeal descent from within the archaeal evolutionary tree, specifically, the TACK superphylum. Fourth, evidence has been presented that the origin of the major archaeal phyla involved massive acquisition of bacterial genes. Taken together, these findings make the symbiogenetic scenario for the origin of eukaryotes considerably more plausible and the origin of the organizational complexity of eukaryotic cells more readily explainable than they appeared until

  10. Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis.

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4-8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes. PMID:25491751

  11. Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis.

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4-8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes.

  12. Molecular Diversity of Eukaryotes in Municipal Wastewater Treatment Processes as Revealed by 18S rRNA Gene Analysis

    PubMed Central

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4–8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes. PMID:25491751

  13. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed

    Periz, G; Keller, L R

    1997-07-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

  14. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed Central

    Periz, G; Keller, L R

    1997-01-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements. PMID:9199320

  15. GC-biased gene conversion impacts ribosomal DNA evolution in vertebrates, angiosperms, and other eukaryotes.

    PubMed

    Escobar, Juan S; Glémin, Sylvain; Galtier, Nicolas

    2011-09-01

    Ribosomal DNA (rDNA) is one of the most conserved genes in eukaryotes. The multiples copies of rDNA in the genome evolve in a concerted manner, through unequal crossing over and/or gene conversion, two mechanisms related to homologous recombination. Recombination increases local GC content in several organisms through a process known as GC-biased gene conversion (gBGC). gBGC has been well characterized in mammals, birds, and grasses, but its phylogenetic distribution across the tree of life is poorly understood. Here, we test the hypothesis that recombination affects the evolution of base composition in 18S rDNA and examine the reliability of this thoroughly studied molecule as a marker of gBGC in eukaryotes. Phylogenetic analyses of 18S rDNA in vertebrates and angiosperms reveal significant heterogeneity in the evolution of base composition across both groups. Mammals, birds, and grasses experience increases in the GC content of the 18S rDNA, consistent with previous genome-wide analyses. In addition, we observe increased GC contents in Ostariophysi ray-finned fishes and commelinid monocots (i.e., the clade including grasses), suggesting that the genomes of these two groups have been affected by gBGC. Polymorphism analyses in rDNA confirm that gBGC, not mutation bias, is the most plausible explanation for these patterns. We also find that helix and loop sites of the secondary structure of ribosomal RNA do not evolve at the same pace: loops evolve faster than helices, whereas helices are GC richer than loops. We extend analyses to major lineages of eukaryotes and suggest that gBGC might have also affected base composition in Giardia (Diplomonadina), nudibranch gastropods (Mollusca), and Asterozoa (Echinodermata). PMID:21444650

  16. A Metastate HMM with Application to Gene Structure Identification in Eukaryotes

    NASA Astrophysics Data System (ADS)

    Winters-Hilt, Stephen; Baribault, Carl

    2010-12-01

    We introduce a generalized-clique hidden Markov model (HMM) and apply it to gene finding in eukaryotes ( C. elegans). We demonstrate a HMM structure identification platform that is novel and robustly-performing in a number of ways. The generalized clique HMM begins by enlarging the primitive hidden states associated with the individual base labels (as exon, intron, or junk) to substrings of primitive hidden states, or footprint states, having a minimal length greater than the footprint state length. The emissions are likewise expanded to higher order in the fundamental joint probability that is the basis of the generalized-clique, or "metastate", HMM. We then consider application to eukaryotic gene finding and show how such a metastate HMM improves the strength of coding/noncoding-transition contributions to gene-structure identification. We will describe situations where the coding/noncoding-transition modeling can effectively recapture the exon and intron heavy tail distribution modeling capability as well as manage the exon-start needle-in-the-haystack problem. In analysis of the C. elegans genome we show that the sensitivity and specificity (SN,SP) results for both the individual-state and full-exon predictions are greatly enhanced over the standard HMM when using the generalized-clique HMM.

  17. GenePainter: a fast tool for aligning gene structures of eukaryotic protein families, visualizing the alignments and mapping gene structures onto protein structures

    PubMed Central

    2013-01-01

    Background All sequenced eukaryotic genomes have been shown to possess at least a few introns. This includes those unicellular organisms, which were previously suspected to be intron-less. Therefore, gene splicing must have been present at least in the last common ancestor of the eukaryotes. To explain the evolution of introns, basically two mutually exclusive concepts have been developed. The introns-early hypothesis says that already the very first protein-coding genes contained introns while the introns-late concept asserts that eukaryotic genes gained introns only after the emergence of the eukaryotic lineage. A very important aspect in this respect is the conservation of intron positions within homologous genes of different taxa. Results GenePainter is a standalone application for mapping gene structure information onto protein multiple sequence alignments. Based on the multiple sequence alignments the gene structures are aligned down to single nucleotides. GenePainter accounts for variable lengths in exons and introns, respects split codons at intron junctions and is able to handle sequencing and assembly errors, which are possible reasons for frame-shifts in exons and gaps in genome assemblies. Thus, even gene structures of considerably divergent proteins can properly be compared, as it is needed in phylogenetic analyses. Conserved intron positions can also be mapped to user-provided protein structures. For their visualization GenePainter provides scripts for the molecular graphics system PyMol. Conclusions GenePainter is a tool to analyse gene structure conservation providing various visualization options. A stable version of GenePainter for all operating systems as well as documentation and example data are available at http://www.motorprotein.de/genepainter.html. PMID:23496949

  18. Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor.

    PubMed

    Yeon, Eun-Hee; Noh, Ju-Young; Kim, Jong-Min; Lee, Min-Young; Yoon, Sarah; Park, Sang-Kyu; Choi, Kang-Yell; Kim, Kyung-Sup

    2004-06-25

    Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or beta-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.

  19. EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases.

    PubMed

    Falk, A; Feys, B J; Frost, L N; Jones, J D; Daniels, M J; Parker, J E

    1999-03-16

    A major class of plant disease resistance (R) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana, EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs (PR1 and PDF1.2, respectively) for these two pathways in wild-type and eds1 mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation and is not required for jasmonic acid-induced PDF1.2 mRNA expression.

  20. Well-characterized sequence features of eukaryote genomes and implications for ab initio gene prediction.

    PubMed

    Huang, Ying; Chen, Shi-Yi; Deng, Feilong

    2016-01-01

    In silico analysis of DNA sequences is an important area of computational biology in the post-genomic era. Over the past two decades, computational approaches for ab initio prediction of gene structure from genome sequence alone have largely facilitated our understanding on a variety of biological questions. Although the computational prediction of protein-coding genes has already been well-established, we are also facing challenges to robustly find the non-coding RNA genes, such as miRNA and lncRNA. Two main aspects of ab initio gene prediction include the computed values for describing sequence features and used algorithm for training the discriminant function, and by which different combinations are employed into various bioinformatic tools. Herein, we briefly review these well-characterized sequence features in eukaryote genomes and applications to ab initio gene prediction. The main purpose of this article is to provide an overview to beginners who aim to develop the related bioinformatic tools. PMID:27536341

  1. The ycf27 genes from cyanobacteria and eukaryotic algae: distribution and implications for chloroplast evolution.

    PubMed

    Ashby, Mark K; Houmard, Jean; Mullineaux, Conrad W

    2002-08-27

    The two ycf27 genes from the filamentous cyanobacterium Tolypothrix PCC 7601 have been cloned and sequenced. These two genes, previously designated rpaA and rpaB, encode putative transcriptional regulators of the 'OmpR' family. In Synechocystis PCC 6803, homologous genes have been linked to the regulation of transfer of excitation energy from the phycobilisome to photosystem (PS) I and PSII respectively. Partial clones from Spirulina platensis, Dactylococcopsis salina and Synechococcus PCC 7002 have also been sequenced. A table of identity between the proteins confirms that RpaB belongs in the same family as the algal ycf27 proteins. However, RpaA is a rather different protein and should lose the designation ycf27. The loss of rpaB from the plastid genomes of eukaryotic algae is associated with the loss of phycobiliproteins, so it is likely that this gene performs a similar role in algae to that in cyanobacteria. The implications for chloroplast evolution are discussed along with the possible identity of the cognate histidine kinase gene in the plastid genomes.

  2. The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution.

    PubMed

    Archibald, John M; O'Kelly, Charles J; Doolittle, W Ford

    2002-04-01

    The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.

  3. Gene fusion, fission, lateral transfer, and loss: Not-so-rare events in the evolution of eukaryotic ATP citrate lyase.

    PubMed

    Gawryluk, Ryan M R; Eme, Laura; Roger, Andrew J

    2015-10-01

    ATP citrate lyase (ACL) is an enzyme critical to the generation of cytosolic acetyl-CoA in eukaryotes. In most studied organisms, ACL activity is conferred in combination by two proteins, ACLA and ACLB (dsACL); however, animals encode a single-subunit ACL (ssACL) - the result of a gene fusion event. Through phylogenetic analyses, we investigated the evolution of ACL in a broad range of eukaryotes, including numerous microbes (protists). We show that the fused form is not restricted to animals, and is instead widely distributed among eukaryotes. Furthermore, ssACL and dsACL are patchily distributed and appear to be mutually exclusive; both types arose early in eukaryotic evolution. Finally, we present several compelling hypotheses of lateral gene transfer and gene loss, along with the secondary gene fission of ssACL in Ascomycota. Collectively, our in-depth analyses suggest that a complex suite of evolutionary events, usually considered rare, has shaped the evolution of ACL in eukaryotes.

  4. The regulation of gene expression in eukaryotes: bistability and oscillations in repressilator models.

    PubMed

    Dilão, Rui

    2014-01-01

    To model the regulation of gene expression in eukaryotes by transcriptional activators and repressors, we introduce delays in conjugation with the mass action law. Delays are associated with the time gap between the mRNA transcription in the nucleoplasm and the protein synthesis in the cytoplasm. After re-parameterisation of the m-repressilator model with the Hill cooperative parameter n, for n=1, the m-repressilator is deducible from the mass action law and, in the limit n→∞, it is a Boolean type model. With this embedding and with delays, if m is odd and n>1, we show that there is always a choice of parameters for which the m-repressilator model has sustained oscillations (limit cycles), implying that the 1-repressilator is the simplest genetic mechanism leading to sustained oscillations in eukaryotes. If m is even and n>1, there is always a choice of parameters for which the m-repressilator model has bistability.

  5. Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria*

    PubMed Central

    Chiba, Yoko; Kamikawa, Ryoma; Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi

    2015-01-01

    Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. PMID:26269598

  6. Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria.

    PubMed

    Chiba, Yoko; Kamikawa, Ryoma; Nakada-Tsukui, Kumiko; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi

    2015-09-25

    Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. PMID:26269598

  7. High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions.

    PubMed

    Hoepfner, Dominic; Helliwell, Stephen B; Sadlish, Heather; Schuierer, Sven; Filipuzzi, Ireos; Brachat, Sophie; Bhullar, Bhupinder; Plikat, Uwe; Abraham, Yann; Altorfer, Marc; Aust, Thomas; Baeriswyl, Lukas; Cerino, Raffaele; Chang, Lena; Estoppey, David; Eichenberger, Juerg; Frederiksen, Mathias; Hartmann, Nicole; Hohendahl, Annika; Knapp, Britta; Krastel, Philipp; Melin, Nicolas; Nigsch, Florian; Oakeley, Edward J; Petitjean, Virginie; Petersen, Frank; Riedl, Ralph; Schmitt, Esther K; Staedtler, Frank; Studer, Christian; Tallarico, John A; Wetzel, Stefan; Fishman, Mark C; Porter, Jeffrey A; Movva, N Rao

    2014-01-01

    Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology. PMID:24360837

  8. The role of horizontal gene transfer in kleptoplastidy and the establishment of photosynthesis in the eukaryotes

    PubMed Central

    Pillet, Loïc

    2013-01-01

    Found in different eukaryotic lineages, kleptoplastidy is the ability to sequester chloroplasts from algal preys that are ingested and partially digested. While most of the genetic information required for the activity and maintenance of the kleptoplastids disappeared with the digestion of the algal nuclei, the photosynthetic organelles remain active during extended period of time. Many different hypotheses have been proposed to explain the longevity of the kleptoplastids within their host. The most popular one involves Horizontal Gene Transfer (HGT) from the algal genome to the host nucleus. In order to test this hypothesis, transcriptome-based analyses have been performed on different kleptoplastidic organisms during the past few years. However, the variability of the results obtained does not allow drawing a convincing conclusion regarding the precise role of HGT in kleptoplastidy. Understanding the mechanism that allow persistence of the plastids is crucial, not only for the characterization of kleptoplastidy, but also for important evolutionary questions surrounding endosymbiotic events and the emergence and spread of photosynthesis in the eukaryotes. Here, I discuss alternative theories that could explain the longevity of sequestered plastids in their host, with special focus on the simplest chloroplast stability hypothesis. PMID:23914312

  9. Metatranscriptomics Reveals the Diversity of Genes Expressed by Eukaryotes in Forest Soils

    PubMed Central

    Damon, Coralie; Lehembre, Frédéric; Oger-Desfeux, Christine; Luis, Patricia; Ranger, Jacques; Fraissinet-Tachet, Laurence; Marmeisse, Roland

    2012-01-01

    Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a

  10. Metatranscriptomics reveals the diversity of genes expressed by eukaryotes in forest soils.

    PubMed

    Damon, Coralie; Lehembre, Frédéric; Oger-Desfeux, Christine; Luis, Patricia; Ranger, Jacques; Fraissinet-Tachet, Laurence; Marmeisse, Roland

    2012-01-01

    Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)-0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a

  11. Evolutionary dynamics of introns in plastid-derived genes in plants: saturation nearly reached but slow intron gain continues.

    PubMed

    Basu, Malay Kumar; Rogozin, Igor B; Deusch, Oliver; Dagan, Tal; Martin, William; Koonin, Eugene V

    2008-01-01

    Some of the principal transitions in the evolution of eukaryotes are characterized by engulfment of prokaryotes by primitive eukaryotic cells. In particular, approximately 1.6 billion years ago, engulfment of a cyanobacterium that became the ancestor of chloroplasts and other plastids gave rise to Plantae, the major branch of eukaryotes comprised of glaucophytes, red algae, green algae, and green plants. After endosymbiosis, there was large-scale migration of genes from the endosymbiont to the nuclear genome of the host such that approximately 18% of the nuclear genes in Arabidopsis appear to be of chloroplast origin. To gain insights into the process of evolution of gene structure in these, originally, intronless genes, we compared the properties and the evolutionary dynamics of introns in genes of plastid origin and ancestral eukaryotic genes in Arabidopsis, poplar, and rice genomes. We found that intron densities in plastid-derived genes were slightly but significantly lower than those in ancestral eukaryotic genes. Although most of the introns in both categories of genes were conserved between monocots (rice) and dicots (Arabidopsis and poplar), lineage-specific intron gain was more pronounced in plastid-derived genes than in ancestral genes, whereas there was no significant difference in the intron loss rates between the 2 classes of genes. Thus, after the transfer to the nuclear genome, the plastid-derived genes have undergone a massive intron invasion that, by the time of the divergence of dicots and monocots (150-200 MYA), yielded intron densities only slightly lower than those in ancestral genes. Nevertheless, the accumulation of introns in plastid-derived genes appears not to have reached saturation and continues to this time, albeit at a low rate. The overall pattern of intron gain and loss in the plastid-derived genes is shaped by this continuing gain and the more general tendency for loss that is characteristic of the recent evolution of plant genes.

  12. The R-Operon: A Model of Repetitive DNA-Organized Transcriptional Compartmentation of Eukaryotic Chromosomes for Coordinated Gene Expression

    PubMed Central

    Tang, Shao-Jun

    2016-01-01

    In eukaryotic genomes, it is essential to coordinate the activity of genes that function together to fulfill the same biological processes. Genomic organization likely plays a key role in coordinating transcription of different genes. However, little is known about how co-regulated genes are organized in the cell nucleus and how the chromosomal organization facilitates the co-regulation of different genes. I propose that eukaryotic genomes are organized into repeat assembly (RA)-based structural domains (“R-operons”) in the nuclear space. R-operons result from the interaction of homologous DNA repeats. In an R-operon, genes in different loci of the linear genome are brought into spatial vicinity and co-regulated by the same pool of transcription factors. This type of large-scale chromosomal organization may provide a mechanism for functional compartmentation of chromosomes to facilitate the transcriptional coordination of gene expression. PMID:27110825

  13. New genes from non-coding sequence: the role of de novo protein-coding genes in eukaryotic evolutionary innovation

    PubMed Central

    McLysaght, Aoife; Guerzoni, Daniele

    2015-01-01

    The origin of novel protein-coding genes de novo was once considered so improbable as to be impossible. In less than a decade, and especially in the last five years, this view has been overturned by extensive evidence from diverse eukaryotic lineages. There is now evidence that this mechanism has contributed a significant number of genes to genomes of organisms as diverse as Saccharomyces, Drosophila, Plasmodium, Arabidopisis and human. From simple beginnings, these genes have in some instances acquired complex structure, regulated expression and important functional roles. New genes are often thought of as dispensable late additions; however, some recent de novo genes in human can play a role in disease. Rather than an extremely rare occurrence, it is now evident that there is a relatively constant trickle of proto-genes released into the testing ground of natural selection. It is currently unknown whether de novo genes arise primarily through an ‘RNA-first’ or ‘ORF-first’ pathway. Either way, evolutionary tinkering with this pool of genetic potential may have been a significant player in the origins of lineage-specific traits and adaptations. PMID:26323763

  14. Eukaryotic cold shock domain proteins: highly versatile regulators of gene expression.

    PubMed

    Mihailovich, Marija; Militti, Cristina; Gabaldón, Toni; Gebauer, Fátima

    2010-02-01

    Cold shock domain (CSD)-containing proteins have been found in all three domains of life and function in a variety of processes that are related, for the most part, to post-transcriptional gene regulation. The CSD is an ancient beta-barrel fold that serves to bind nucleic acids. The CSD is structurally and functionally similar to the S1 domain, a fold with otherwise unrelated primary sequence. The flexibility of the CSD/S1 domain for RNA recognition confers an enormous functional versatility to the proteins that contain them. This review summarizes the current knowledge on eukaryotic CSD/S1 domain-containing proteins with a special emphasis on UNR (upstream of N-ras), a member of this family with multiple copies of the CSD.

  15. The Ccr4‐Not complex is a key regulator of eukaryotic gene expression

    PubMed Central

    2016-01-01

    The Ccr4‐Not complex is a multisubunit complex present in all eukaryotes that contributes to regulate gene expression at all steps, from production of messenger RNAs (mRNAs) in the nucleus to their degradation in the cytoplasm. In the nucleus it influences the post‐translational modifications of the chromatin template that has to be remodeled for transcription, it is present at sites of transcription and associates with transcription factors as well as with the elongating polymerase, it interacts with the factors that prepare the new transcript for export to the cytoplasm and finally is important for nuclear quality control and influences mRNA export. In the cytoplasm it is present in polysomes where mRNAs are translated and in RNA granules where mRNAs will be redirected upon inhibition of translation. It influences mRNA translatability, and is needed during translation, on one hand for co‐translational protein interactions and on the other hand to preserve translation that stalls. It is one of the relevant players during co‐translational quality control. It also interacts with factors that will repress translation or induce mRNA decapping when recruited to the translating template. Finally, Ccr4‐Not carries deadenylating enzymes and is a key player in mRNA decay, generic mRNA decay that follows normal translation termination, co‐translational mRNA decay of transcripts on which the ribosomes stall durably or which carry a non‐sense mutation and finally mRNA decay that is induced by external signaling for a change in genetic programming. Ccr4‐Not is a master regulator of eukaryotic gene expression. WIREs RNA 2016, 7:438–454. doi: 10.1002/wrna.1332 For further resources related to this article, please visit the WIREs website. PMID:26821858

  16. Statistical analysis of the exon-intron structure of higher and lower eukaryote genes.

    PubMed

    Kriventseva, E V; Gelfand, M S

    1999-10-01

    Statistics of the exon-intron structure and splicing sites of several diverse eukaryotes was studied. The yeast exon-intron structures have a number of unique features. A yeast gene usually have at most one intron. The branch site is strongly conserved, whereas the polypirimidine tract is short. Long yeast introns tend to have stronger acceptor sites. In other species the branch site is less conserved and often cannot be determined. In non-yeast samples there is an almost universal correlation between lengths of neighboring exons (all samples excluding protists) and correlation between lengths of neighboring introns (human, drosophila, protists). On the average first introns are longer, and anomalously long introns are usually first introns in a gene. There is a universal preference for exons and exon pairs with the (total) length divisible by 3. Introns positioned between codons are preferred, whereas those positioned between the first and second positions in codon are avoided. The choice of A or G at the third position of intron (the donor splice sites generally prefer purines at this position) is correlated with the overall GC-composition of the gene. In all samples dinucleotide AG is avoided in the region preceding the acceptor site.

  17. Horizontal gene transfer in eukaryotic parasites: a case study of Entamoeba histolytica and Trichomonas vaginalis.

    PubMed

    Alsmark, U Cecilia; Sicheritz-Ponten, Thomas; Foster, Peter G; Hirt, Robert P; Embley, T Martin

    2009-01-01

    Over the past few years it has become apparent that horizontal gene transfer (HGT) has played an important role in the evolution of pathogenic prokaryotes. What is less clear is the exact role that HGT has played in shaping the metabolism of eukaryotic organisms. The main problems are the reliable inference of HGT on a genomic scale as well as the functional assignment of genes in these poorly studied organisms. We have screened the completed genomes of the protists Entamoeba histolytica and Trichomonas vaginalis for cases of HGT from prokaryotes. Using a fast primary screen followed by a conservative phylogenetic approach, we found 68 and 153 recent cases of HGT in the respective organisms. The majority of transferred genes that fall into functional categories code for enzymes involved in metabolism. We found a broad range of prokaryotic lineages represented among the donors, but organisms that share similar environmental niches with E. histolytica and T. vaginalis, such as the gut and the vaginal mucosa, dominate.

  18. FrameD: a flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences

    PubMed Central

    Schiex, Thomas; Gouzy, Jérôme; Moisan, Annick; de Oliveira, Yannick

    2003-01-01

    We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms. PMID:12824407

  19. FrameD: A flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences.

    PubMed

    Schiex, Thomas; Gouzy, Jérôme; Moisan, Annick; de Oliveira, Yannick

    2003-07-01

    We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms. PMID:12824407

  20. Genomic distribution of AFLP markers relative to gene locations for different eukaryotic species

    PubMed Central

    2013-01-01

    Background Amplified fragment length polymorphism (AFLP) markers are frequently used for a wide range of studies, such as genome-wide mapping, population genetic diversity estimation, hybridization and introgression studies, phylogenetic analyses, and detection of signatures of selection. An important issue to be addressed for some of these fields is the distribution of the markers across the genome, particularly in relation to gene sequences. Results Using in-silico restriction fragment analysis of the genomes of nine eukaryotic species we characterise the distribution of AFLP fragments across the genome and, particularly, in relation to gene locations. First, we identify the physical position of markers across the chromosomes of all species. An observed accumulation of fragments around (peri) centromeric regions in some species is produced by repeated sequences, and this accumulation disappears when AFLP bands rather than fragments are considered. Second, we calculate the percentage of AFLP markers positioned within gene sequences. For the typical EcoRI/MseI enzyme pair, this ranges between 28 and 87% and is usually larger than that expected by chance because of the higher GC content of gene sequences relative to intergenic ones. In agreement with this, the use of enzyme pairs with GC-rich restriction sites substantially increases the above percentages. For example, using the enzyme system SacI/HpaII, 86% of AFLP markers are located within gene sequences in A. thaliana, and 100% of markers in Plasmodium falciparun. We further find that for a typical trait controlled by 50 genes of average size, if 1000 AFLPs are used in a study, the number of those within 1 kb distance from any of the genes would be only about 1–2, and only about 50% of the genes would have markers within that distance. Conclusions The high coverage of AFLP markers across the genomes and the high proportion of markers within or close to gene sequences make them suitable for genome scans and

  1. Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

    PubMed Central

    Novo, Maite; Bigey, Frédéric; Beyne, Emmanuelle; Galeote, Virginie; Gavory, Frédérick; Mallet, Sandrine; Cambon, Brigitte; Legras, Jean-Luc; Wincker, Patrick; Casaregola, Serge; Dequin, Sylvie

    2009-01-01

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. PMID:19805302

  2. Comprehensive analysis of amino acid and nucleotide composition in eukaryotic genomes, comparing genes and pseudogenes.

    PubMed

    Echols, Nathaniel; Harrison, Paul; Balasubramanian, Suganthi; Luscombe, Nicholas M; Bertone, Paul; Zhang, Zhaolei; Gerstein, Mark

    2002-06-01

    Based on searches for disabled homologs to known proteins, we have identified a large population of pseudogenes in four sequenced eukaryotic genomes-the worm, yeast, fly and human (chromosomes 21 and 22 only). Each of our nearly 2500 pseudogenes is characterized by one or more disablements mid-domain, such as premature stops and frameshifts. Here, we perform a comprehensive survey of the amino acid and nucleotide composition of these pseudogenes in comparison to that of functional genes and intergenic DNA. We show that pseudogenes invariably have an amino acid composition intermediate between genes and translated intergenic DNA. Although the degree of intermediacy varies among the four organisms, in all cases, it is most evident for amino acid types that differ most in occurrence between genes and intergenic regions. The same intermediacy also applies to codon frequencies, especially in the worm and human. Moreover, the intermediate composition of pseudogenes applies even though the composition of the genes in the four organisms is markedly different, showing a strong correlation with the overall A/T content of the genomic sequence. Pseudogenes can be divided into 'ancient' and 'modern' subsets, based on the level of sequence identity with their closest matching homolog (within the same genome). Modern pseudogenes usually have a much closer sequence composition to genes than ancient pseudogenes. Collectively, our results indicate that the composition of pseudogenes that are under no selective constraints progressively drifts from that of coding DNA towards non-coding DNA. Therefore, we propose that the degree to which pseudogenes approach a random sequence composition may be useful in dating different sets of pseudogenes, as well as to assess the rate at which intergenic DNA accumulates mutations. Our compositional analyses with the interactive viewer are available over the web at http://genecensus.org/pseudogene.

  3. Split-Doa10: A Naturally Split Polytopic Eukaryotic Membrane Protein Generated by Fission of a Nuclear Gene

    PubMed Central

    Stuerner, Elisabeth; Kuraku, Shigehiro; Hochstrasser, Mark; Kreft, Stefan G.

    2012-01-01

    Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12–16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments. PMID:23071509

  4. Ranking Gene Ontology terms for predicting non-classical secretory proteins in eukaryotes and prokaryotes.

    PubMed

    Huang, Wen-Lin

    2012-11-01

    Protein secretion is an important biological process for both eukaryotes and prokaryotes. Several sequence-based methods mainly rely on utilizing various types of complementary features to design accurate classifiers for predicting non-classical secretory proteins. Gene Ontology (GO) terms are increasing informative in predicting protein functions. However, the number of used GO terms is often very large. For example, there are 60,020 GO terms used in the prediction method Euk-mPLoc 2.0 for subcellular localization. This study proposes a novel approach to identify a small set of m top-ranked GO terms served as the only type of input features to design a support vector machine (SVM) based method Sec-GO to predict non-classical secretory proteins in both eukaryotes and prokaryotes. To evaluate the Sec-GO method, two existing methods and their used datasets are adopted for performance comparisons. The Sec-GO method using m=436 GO terms yields an independent test accuracy of 96.7% on mammalian proteins, much better than the existing method SPRED (82.2%) which uses frequencies of tri-peptides and short peptides, secondary structure, and physicochemical properties as input features of a random forest classifier. Furthermore, when applying to Gram-positive bacterial proteins, the Sec-GO with m=158 GO terms has a test accuracy of 94.5%, superior to NClassG+ (90.0%) which uses SVM with several feature types, comprising amino acid composition, di-peptides, physicochemical properties and the position specific weighting matrix. Analysis of the distribution of secretory proteins in a GO database indicates the percentage of the non-classical secretory proteins annotated by GO is larger than that of classical secretory proteins in both eukaryotes and prokaryotes. Of the m top-ranked GO features, the top-four GO terms are all annotated by such subcellular locations as GO:0005576 (Extracellular region). Additionally, the method Sec-GO is easily implemented and its web tool of

  5. Evolution of PAS domains and PAS-containing genes in eukaryotes.

    PubMed

    Mei, Qiming; Dvornyk, Volodymyr

    2014-08-01

    The PAS domains are signal modules, which are widely distributed in proteins across all kingdoms of life. They are common in photoreceptors and transcriptional regulators of eukaryotic circadian clocks q(bHLH-PAS proteins and PER in animals; PHY and ZTL in plants; and WC-1, 2, and VVD in fungi) and possess mainly protein-protein interaction and light-sensing functions. We conducted several evolutionary analyses of the PAS superfamily. Although the whole superfamily evolved primarily under strong purifying selection (average ω ranges from 0.0030 to 0.1164), some lineages apparently experienced strong episodic positive selection at some periods of the evolution. Although the PAS domains from different proteins vary in sequence and length, but they maintain a fairly conserved 3D structure, which is determined by only eight residues. The WC-1 and WC- 2, bHLH-PAS, and P er genes probably originated in the Neoproterozoic Era (1000-542 Mya), plant P hy and ZTL evolved in the Paleozoic (541-252 Mya), which might be a result of adaptation to the major climate and global light regime changes having occurred in those eras.

  6. Horizontal DNA transfer from bacteria to eukaryotes and a lesson from experimental transfers.

    PubMed

    Suzuki, Katsunori; Moriguchi, Kazuki; Yamamoto, Shinji

    2015-12-01

    Horizontal gene transfer (HGT) is widespread among bacteria and plays a key role in genome dynamics. HGT is much less common in eukaryotes, but is being reported with increasing frequency in eukaryotes. The mechanism as to how eukaryotes acquired genes from distantly related organisms remains obscure yet. This paper cites examples of bacteria-derived genes found in eukaryotic organisms, and then describes experimental DNA transports to eukaryotes by bacterial type 4 secretion systems in optimized conditions. The mechanisms of the latter are efficient, quite reproducible in vitro and predictable, and thereby would provide insight into natural HGT and to the development of new research tools.

  7. topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

    PubMed Central

    Dahmane, Narimane; Gadelle, Danièle; Delmas, Stéphane; Criscuolo, Alexis; Eberhard, Stephan; Desnoues, Nicole; Collin, Sylvie; Zhang, Hongliang; Pommier, Yves; Forterre, Patrick; Sezonov, Guennadi

    2016-01-01

    Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumarchaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Nitrososphaera viennensis and we purified the recombinant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described thermostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies. PMID:26908651

  8. topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB.

    PubMed

    Dahmane, Narimane; Gadelle, Danièle; Delmas, Stéphane; Criscuolo, Alexis; Eberhard, Stephan; Desnoues, Nicole; Collin, Sylvie; Zhang, Hongliang; Pommier, Yves; Forterre, Patrick; Sezonov, Guennadi

    2016-04-01

    Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumarchaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Nitrososphaera viennensis and we purified the recombinant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described thermostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies. PMID:26908651

  9. UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan

    PubMed Central

    Ghosh, Arpan C.; Shimell, MaryJane; Leof, Emma R.; Haley, Macy J.; O’Connor, Michael B.

    2015-01-01

    Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage pathway enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP). The enzyme is highly conserved from prokaryotes to humans and yet phylogenetic evidence suggests that UPRT homologues from higher-eukaryotes, including Drosophila, are incapable of binding uracil. Purified human UPRT also do not show any enzymatic activity in vitro, making microbial UPRT an attractive candidate for anti-microbial drug development, suicide-gene therapy, and cell-specific mRNA labeling techniques. Nevertheless, the enzymatic site of UPRT remains conserved across the animal kingdom indicating an in vivo role for the enzyme. We find that the Drosophila UPRT homologue, krishah (kri), codes for an enzyme that is required for larval growth, pre-pupal/pupal viability and long-term adult lifespan. Our findings suggest that UPRT from all higher eukaryotes is likely enzymatically active in vivo and challenges the previous notion that the enzyme is non-essential in higher eukaryotes and cautions against targeting the enzyme for therapeutic purposes. Our findings also suggest that expression of the endogenous UPRT gene will likely cause background incorporation when using microbial UPRT as a cell-specific mRNA labeling reagent in higher eukaryotes. PMID:26271729

  10. Effect of antisense MBD1 gene eukaryotic expression plasmid on expression of MBD1 gene in human biliary tract carcinoma cells.

    PubMed

    Zuo, Shi; Zou, Shengquan; Luo, Jian; Guo, Wei; Xu, Lining; Dong, Jingqing; Liu, Minfeng

    2005-01-01

    Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma. PMID:16696318

  11. Parallel re-modeling of EF-1α function: divergent EF-1α genes co-occur with EFL genes in diverse distantly related eukaryotes

    PubMed Central

    2013-01-01

    Background Elongation factor-1α (EF-1α) and elongation factor-like (EFL) proteins are functionally homologous to one another, and are core components of the eukaryotic translation machinery. The patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a ‘differential loss’ hypothesis that assumes that EF-1α and EFL were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. To date, however, just one diatom and one fungus have been found to have both EF-1α and EFL (dual-EF-containing species). Results In this study, we characterized 35 new EF-1α/EFL sequences from phylogenetically diverse eukaryotes. In so doing we identified 11 previously unreported dual-EF-containing species from diverse eukaryote groups including the Stramenopiles, Apusomonadida, Goniomonadida, and Fungi. Phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-EF lineages. In the dual-EF-containing species we identified, the EF-1α genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring EFL genes. Conclusions According to the known EF-1α/EFL distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-EF-containing species remain unidentified. We predict that dual-EF-containing species retain the divergent EF-1α homologues only for a sub-set of the original functions. As the dual-EF-containing species are distantly related to each other, we propose that independent re-modelling of EF-1α function took place in multiple branches in the tree of eukaryotes. PMID:23800323

  12. Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote.

    PubMed

    Heller, Jens; Zhao, Jiuhai; Rosenfield, Gabriel; Kowbel, David J; Gladieux, Pierre; Glass, N Louise

    2016-04-01

    Microorganisms are capable of communication and cooperation to perform social activities. Cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. In the filamentous fungus Neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. Recognition and chemotropic interactions between isogenic germlings requires oscillation of the mitogen-activated protein kinase (MAPK) signal transduction protein complex (NRC-1, MEK-2, MAK-2, and the scaffold protein HAM-5) to specialized cell fusion structures termed conidial anastomosis tubes. Using a population of 110 wild N. crassa isolates, we investigated germling fusion between genetically unrelated individuals and discovered that chemotropic interactions are regulated by kind discrimination. Distinct communication groups were identified, in which germlings within one communication group interacted at high frequency, while germlings from different communication groups avoided each other. Bulk segregant analysis followed by whole genome resequencing identified three linked genes (doc-1, doc-2, and doc-3), which were associated with communication group phenotype. Alleles at doc-1, doc-2, and doc-3 fell into five haplotypes that showed transspecies polymorphism. Swapping doc-1 and doc-2 alleles from different communication group strains was necessary and sufficient to confer communication group affiliation. During chemotropic interactions, DOC-1 oscillated with MAK-2 to the tips of conidial anastomosis tubes, while DOC-2 was statically localized to the plasma membrane. Our data indicate that doc-1, doc-2, and doc-3 function as "greenbeard" genes, involved in mediating long-distance kind recognition that involves actively searching for one's own type, resulting in cooperation between

  13. Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote

    PubMed Central

    Heller, Jens; Zhao, Jiuhai; Rosenfield, Gabriel; Kowbel, David J.; Gladieux, Pierre; Glass, N. Louise

    2016-01-01

    Microorganisms are capable of communication and cooperation to perform social activities. Cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. In the filamentous fungus Neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. Recognition and chemotropic interactions between isogenic germlings requires oscillation of the mitogen-activated protein kinase (MAPK) signal transduction protein complex (NRC-1, MEK-2, MAK-2, and the scaffold protein HAM-5) to specialized cell fusion structures termed conidial anastomosis tubes. Using a population of 110 wild N. crassa isolates, we investigated germling fusion between genetically unrelated individuals and discovered that chemotropic interactions are regulated by kind discrimination. Distinct communication groups were identified, in which germlings within one communication group interacted at high frequency, while germlings from different communication groups avoided each other. Bulk segregant analysis followed by whole genome resequencing identified three linked genes (doc-1, doc-2, and doc-3), which were associated with communication group phenotype. Alleles at doc-1, doc-2, and doc-3 fell into five haplotypes that showed transspecies polymorphism. Swapping doc-1 and doc-2 alleles from different communication group strains was necessary and sufficient to confer communication group affiliation. During chemotropic interactions, DOC-1 oscillated with MAK-2 to the tips of conidial anastomosis tubes, while DOC-2 was statically localized to the plasma membrane. Our data indicate that doc-1, doc-2, and doc-3 function as “greenbeard” genes, involved in mediating long-distance kind recognition that involves actively searching for one’s own type, resulting in cooperation

  14. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    PubMed

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  15. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources

    PubMed Central

    Stanke, Mario; Schöffmann, Oliver; Morgenstern, Burkhard; Waack, Stephan

    2006-01-01

    Background In order to improve gene prediction, extrinsic evidence on the gene structure can be collected from various sources of information such as genome-genome comparisons and EST and protein alignments. However, such evidence is often incomplete and usually uncertain. The extrinsic evidence is usually not sufficient to recover the complete gene structure of all genes completely and the available evidence is often unreliable. Therefore extrinsic evidence is most valuable when it is balanced with sequence-intrinsic evidence. Results We present a fairly general method for integration of external information. Our method is based on the evaluation of hints to potentially protein-coding regions by means of a Generalized Hidden Markov Model (GHMM) that takes both intrinsic and extrinsic information into account. We used this method to extend the ab initio gene prediction program AUGUSTUS to a versatile tool that we call AUGUSTUS+. In this study, we focus on hints derived from matches to an EST or protein database, but our approach can be used to include arbitrary user-defined hints. Our method is only moderately effected by the length of a database match. Further, it exploits the information that can be derived from the absence of such matches. As a special case, AUGUSTUS+ can predict genes under user-defined constraints, e.g. if the positions of certain exons are known. With hints from EST and protein databases, our new approach was able to predict 89% of the exons in human chromosome 22 correctly. Conclusion Sensitive probabilistic modeling of extrinsic evidence such as sequence database matches can increase gene prediction accuracy. When a match of a sequence interval to an EST or protein sequence is used it should be treated as compound information rather than as information about individual positions. PMID:16469098

  16. Experimental examination of EFL and MATX eukaryotic horizontal gene transfers: coexistence of mutually exclusive transcripts predates functional rescue.

    PubMed

    Szabová, Jana; Ruzicka, Petr; Verner, Zdenek; Hampl, Vladimír; Lukes, Julius

    2011-08-01

    Many eukaryotic genes do not follow simple vertical inheritance. Elongation factor 1α (EF-1α) and methionine adenosyl transferase (MAT) are enzymes with complicated evolutionary histories and, interestingly, the two cases have several features in common. These essential enzymes occur as two relatively divergent paralogs (EF-1α/EFL, MAT/MATX) that have patchy distributions in eukaryotic lineages that are nearly mutually exclusive. To explain such distributions, we must invoke either multiple eukaryote-to-eukaryote horizontal gene transfers (HGTs) followed by functional replacement or presence of both paralogs in the common ancestor followed by long-term coexistence and differential losses in various eukaryotic lineages. To understand the evolution of these paralogs, we have performed in vivo experiments in Trypanosoma brucei addressing the consequences of long-term coexpression and functional replacement. In the first experiment of its kind, we have demonstrated that EF-1α and MAT can be simultaneously expressed with EFL and MATX, respectively, without affecting the growth of the flagellates. After the endogenous MAT or EF-1α was downregulated by RNA interference, MATX immediately substituted for its paralog, whereas EFL was not able to substitute for EF-1α, leading to mortality. We conclude that MATX is naturally capable of evolving patchy paralog distribution via HGTs and/or long- term coexpression and differential losses. The capability of EFL to spread by HGT is lower and so the patchy distribution of EF-1α/EFL paralogs was probably shaped mainly by deep paralogy followed by long-term coexistence and differential losses.

  17. Reconstructing Early Events in Eukaryotic Evolution.

    PubMed

    Roger

    1999-10-01

    Resolving the order of events that occurred during the transition from prokaryotic to eukaryotic cells remains one of the greatest problems in cell evolution. One view, the Archezoa hypothesis, proposes that the endosymbiotic origin of mitochondria occurred relatively late in eukaryotic evolution and that several mitochondrion-lacking protist groups diverged before the establishment of the organelle. Phylogenies based on small subunit ribosomal RNA and several protein-coding genes supported this proposal, placing amitochondriate protists such as diplomonads, parabasalids, and Microsporidia as the earliest diverging eukaryotic lineages. However, trees of other molecules, such as tubulins, heat shock protein 70, TATA box-binding protein, and the largest subunit of RNA polymerase II, indicate that Microsporidia are not deeply branching eukaryotes but instead are close relatives of the Fungi. Furthermore, recent discoveries of mitochondrion-derived genes in the nuclear genomes of entamoebae, Microsporidia, parabasalids, and diplomonads suggest that these organisms likely descend from mitochondrion-bearing ancestors. Although several protist lineages formally remain as candidates for Archezoa, most evidence suggests that the mitochondrial endosymbiosis took place prior to the divergence of all extant eukaryotes. In addition, discoveries of proteobacterial-like nuclear genes coding for cytoplasmic proteins indicate that the mitochondrial symbiont may have contributed more to the eukaryotic lineage than previously thought. As genome sequence data from parabasalids and diplomonads accumulate, it is becoming clear that the last common ancestor of these protist taxa and other extant eukaryotic groups already possessed many of the complex features found in most eukaryotes but lacking in prokaryotes. However, our confidence in the deeply branching position of diplomonads and parabasalids among eukaryotes is weakened by conflicting phylogenies and potential sources of artifact

  18. Analyses of RNA Polymerase II genes from free-living protists: phylogeny, long branch attraction, and the eukaryotic big bang.

    PubMed

    Dacks, Joel B; Marinets, Alexandra; Ford Doolittle, W; Cavalier-Smith, Thomas; Logsdon, John M

    2002-06-01

    The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist

  19. Centromeres were derived from telomeres during the evolution of the eukaryotic chromosome

    PubMed Central

    Villasante, Alfredo; Abad, José P.; Méndez-Lago, María

    2007-01-01

    The centromere is the DNA region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure the segregation of chromatids during mitosis and of homologous chromosomes in meiosis. The origin of centromeres, therefore, is inseparable from the evolution of cytoskeletal components that distribute chromosomes to offspring cells. Although the origin of the nucleus has been debated, no explanation for the evolutionary appearance of centromeres is available. We propose an evolutionary scenario: The centromeres originated from telomeres. The breakage of the ancestral circular genophore activated the transposition of retroelements at DNA ends that allowed the formation of telomeres by a recombination-dependent replication mechanism. Afterward, the modification of the tubulin-based cytoskeleton that allowed specific subtelomeric repeats to be recognized as new cargo gave rise to the first centromere. This switch from actin-based genophore partition to a tubulin-based mechanism generated a transition period during which both types of cytoskeleton contributed to fidelity of chromosome segregation. During the transition, pseudodicentric chromosomes increased the tendency toward chromosomal breakage and instability. This instability generated multiple telocentric chromosomes that eventually evolved into metacentric or holocentric chromosomes. PMID:17557836

  20. Molecular characterization and phylogenetic analysis of the eukaryotic translation initiation factor 4A gene in Antheraea pernyi (Lepdoptera: Saturniidae).

    PubMed

    Chen, Miao-Miao; Liu, Yan-Qun; Li, Yan; Yao, Rui; Chen, Mo; Xia, Run-Xi; Li, Qun; Qin, Li

    2014-01-01

    Eukaryotic initiation factor 4A (eIF-4A) is an essential component for protein translation in eukaryotes. The eIF-4A gene (ApeIF-4A) was isolated and characterized from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae). The obtained cDNA sequence was 1,435-bp long with an open reading frame of 1,266 bp encoding 421 amino acids. The predicted amino acid sequence shared several conserved features as found in known eIF-4As and revealed 74 and 78% identities with eIF-4As of Homo sapiens L. and Drosophila melanogaster (Meigen), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that ApeIF-4A was transcribed at four developmental stages and in all tissues tested, suggesting that it plays an important role in development of A. pernyi. Homologous alignment suggested that eIF-4As are highly conserved throughout evolution of eukaryote organisms. Phylogenetic trees based on the amino acid and nucleotide sequences of eIF-4A demonstrated a similar topology with the classical systematics, suggesting that it has the potential value in phylogenetic inference of eukaryotes.

  1. The maximal C(3) self-complementary trinucleotide circular code X in genes of bacteria, eukaryotes, plasmids and viruses.

    PubMed

    Michel, Christian J

    2015-09-01

    In 1996, a set X of 20 trinucleotides is identified in genes of both prokaryotes and eukaryotes which has in average the highest occurrence in reading frame compared to the two shifted frames (Arquès and Michel, 1996). Furthermore, this set X has an interesting mathematical property as X is a maximal C(3) self-complementary trinucleotide circular code (Arquès and Michel, 1996). In 2014, the number of trinucleotides in prokaryotic genes has been multiplied by a factor of 527. Furthermore, two new gene kingdoms of plasmids and viruses contain enough trinucleotide data to be analysed. The approach used in 1996 for identifying a preferential frame for a trinucleotide is quantified here with a new definition analysing the occurrence probability of a complementary/permutation (CP) trinucleotide set in a gene kingdom. Furthermore, in order to increase the statistical significance of results compared to those of 1996, the circular code X is studied on several gene taxonomic groups in a kingdom. Based on this new statistical approach, the circular code X is strengthened in genes of prokaryotes and eukaryotes, and now also identified in genes of plasmids. A subset of X with 18 or 16 trinucleotides is identified in genes of viruses. Furthermore, a simple probabilistic model based on the independent occurrence of trinucleotides in reading frame of genes explains the circular code frequencies and asymmetries observed in the shifted frames in all studied gene kingdoms. Finally, the developed approach allows to identify variant X codes in genes, i.e. trinucleotide codes which differ from X. In genes of bacteria, eukaryotes and plasmids, 14 among the 47 studied gene taxonomic groups (about 30%) have variant X codes. Seven variant X codes are identified with at least 16 trinucleotides of X. Two variant X codes XA in cyanobacteria and plasmids of cyanobacteria, and XD in birds are self-complementary, without permuted trinucleotides but non-circular. Five variant X codes XB in

  2. Automatic gene collection system for genome-scale overview of G-protein coupled receptors in eukaryotes.

    PubMed

    Ono, Yukiteru; Fujibuchi, Wataru; Suwa, Makiko

    2005-12-30

    We have developed an automatic system for identifying GPCR (G-protein coupled receptor) genes from various kinds of genomes, which is finally deposited in the SEVENS database (http://sevens.cbrc.jp/), by integrating such software as a gene finder, a sequence alignment tool, a motif and domain assignment tool, and a transmembrane helix predictor. SEVENS enables us to perform a genome-scale overview of the "GPCR universe" using sequences that are identified with high accuracy (99.4% sensitivity and 96.6% specificity). Using this system, we surveyed the complete genomes of 7 eukaryotes and 224 prokaryotes, and found that there are 4 to 1016 GPCR genes in the 7 eukaryotes, and only a total of 16 GPCR genes in all the prokaryotes. Our preliminary results indicate that 11 subfamilies of the Class A family, the Class 2(B) family, the Class 3(C) family and the fz/smo family are commonly found among human, fly, and nematode genomes. We also analyzed the chromosomal locations of the GPCR genes with the Kolmogorov-Smirnov test, and found that species-specific families, such as olfactory, taste, and chemokine receptors in human and nematode chemoreceptor in worm, tend to form clusters extensively, whereas no significant clusters were detected in fly and plant genomes.

  3. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote

    PubMed Central

    Schübeler, Dirk; MacAlpine, David M.; Scalzo, David; Wirbelauer, Christiane; Kooperberg, Charles; van Leeuwen, Fred; Gottschling, Daniel E.; O'Neill, Laura P.; Turner, Bryan M.; Delrow, Jeffrey; Bell, Stephen P.; Groudine, Mark

    2004-01-01

    The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity. PMID:15175259

  4. A Narf-like gene from Cryptosporidium parvum resembles homologues observed in aerobic protists and higher eukaryotes.

    PubMed

    Stejskal, Frantisek; Slapeta, Jan; Ctrnáctá, Vlasta; Keithly, Janet S

    2003-12-01

    Here we report a Narf-like gene from the apicomplexan Cryptosporidium parvum (CpNARF). CpNARF is an intronless, single-copy gene of 1680 bp which encodes a putative protein of 560 amino acids with a calculated molecular mass of 63.1 kDa. This gene contains a single highly conserved N-terminal iron-sulfur cluster ([4Fe-4S]) binding site, as well as most of the H-cluster conserved residues. Reverse transcription polymerase chain reaction analysis indicates that CpNARF is expressed by the intracellular stages of C. parvum. Although the function of this gene is as yet unknown, phylogenetic analyses suggest that CpNARF belongs to the group of NARF-like proteins from aerobic protists and higher eukaryotes, which are thought to have had an ancestor in common with [Fe]-hydrogenases.

  5. Polyphyly of nuclear lamin genes indicates an early eukaryotic origin of the metazoan-type intermediate filament proteins

    PubMed Central

    Kollmar, Martin

    2015-01-01

    The nuclear lamina is a protein meshwork associated with the inner side of the nuclear envelope contributing structural, signalling and regulatory functions. Here, I report on the evolution of an important component of the lamina, the lamin intermediate filament proteins, across the eukaryotic tree of life. The lamins show a variety of protein domain and sequence motif architectures beyond the classical α-helical rod, nuclear localisation signal, immunoglobulin domain and CaaX motif organisation, suggesting extension and adaptation of functions in many species. I identified lamin genes not only in metazoa and Amoebozoa as previously described, but also in other opisthokonts including Ichthyosporea and choanoflagellates, in oomycetes, a sub-family of Stramenopiles, and in Rhizaria, implying that they must have been present very early in eukaryotic evolution if not even the last common ancestor of all extant eukaryotes. These data considerably extend the current perception of lamin evolution and have important implications with regard to the evolution of the nuclear envelope. PMID:26024016

  6. Uptake and distribution of haematoporphyrin derivative in the unicellular eukaryote Paramecium.

    PubMed

    Croce, A C; Wyroba, E; Bottiroli, G

    1990-08-01

    Haematoporphyrin derivative (HpD) uptake, intracellular distribution and turnover were examined in a free-living protozoan cell, Paramecium aurelia, which had been demonstrated to internalize lipoproteins. A 10 min incubation in HpD completed with low-density lipoprotein (LDL) resulted in the appearance of distinct red-fluorescing vesicles, together with a diffuse fluorescence of the cytoplasm. Fluorescein labelling of LDL demonstrated the intracellular localization of HpD and LDL within the same vesicles. Pretreatment of Paramecium with the β-adrenergic antagonist l-propranolol, which blocked its phagocytotic activity, resulted in an absence of red-fluorescing vesicles; thus these were proved to be endosomes. Fluorescence emission recorded in the endosomes was characterized by a band at about 660-670 nm which was attributed to the partially unfolded oligomers; this emission was present during maintenance of the cells in drug-free culture medium for up to 120 min. Propranolol-pretreated cells exhibited only a diffuse cytoplasmic fluorescence characterized by an emission band at 630 nm, which was attributed to the monomers; this disappeared rapidly on washing. These results suggest the following: (i) HpD monomers enter Paramecium via transmembrane influx and/or fluid phase uptake; (ii) HpD oligomers are mainly internalized via receptor-mediated endocytosis; (iii) the extent of the endocytotic process is increased when HpD is completed with LDL; (iv) after internalization, aggregate species undergo a disaggregating process which accounts for the persistence of the intracellular fluorescence.

  7. [Genomic noncoding sequences and the size of eukaryotic cell nucleus as important factors of gene protection from chemical mutagens].

    PubMed

    Minkevich, I G; Patrushev, L I

    2007-01-01

    An improved quantitative model describing a protective function of eukaryotic genomic noncoding sequences was developed. In this new model, two factors affecting gene protection from chemical mutagens are considered: (1) the ratio of the total lengths of coding and noncoding genomic sequences and (2) the volume of the cell nucleus. An increase in the noncoding DNA in the genome reduces the number of mutagen-damaged nucleotides in the coding region, whereas an increase in the volume of the nucleus decreases the flow of mutagens per unit of nuclear volume that attacks its surface.

  8. A metagenome for lacustrine Cladophora (Cladophorales) reveals remarkable diversity of eukaryotic epibionts and genes relevant to materials cycling.

    PubMed

    Graham, Linda E; Knack, Jennifer J; Graham, Melissa E; Graham, James M; Zulkifly, Shahrizim

    2015-06-01

    Periphyton dominated by the cellulose-rich filamentous green alga Cladophora forms conspicuous growths along rocky marine and freshwater shorelines worldwide, providing habitat for diverse epibionts. Bacterial epibionts have been inferred to display diverse functions of biogeochemical significance: N-fixation and other redox reactions, phosphorus accumulation, and organic degradation. Here, we report taxonomic diversity of eukaryotic and prokaryotic epibionts and diversity of genes associated with materials cycling in a Cladophora metagenome sampled from Lake Mendota, Dane Co., WI, USA, during the growing season of 2012. A total of 1,060 distinct 16S, 173 18S, and 351 28S rRNA operational taxonomic units, from which >220 genera or species of bacteria (~60), protists (~80), fungi (6), and microscopic metazoa (~80), were distinguished with the use of reference databases. We inferred the presence of several algal taxa generally associated with marine systems and detected Jaoa, a freshwater periphytic ulvophyte previously thought endemic to China. We identified six distinct nifH gene sequences marking nitrogen fixation, >25 bacterial and eukaryotic cellulases relevant to sedimentary C-cycling and technological applications, and genes encoding enzymes in aerobic and anaerobic pathways for vitamin B12 biosynthesis. These results emphasize the importance of Cladophora in providing habitat for microscopic metazoa, fungi, protists, and bacteria that are often inconspicuous, yet play important roles in ecosystem biogeochemistry.

  9. Predicting protein function with hierarchical phylogenetic profiles: the Gene3D Phylo-Tuner method applied to eukaryotic genomes.

    PubMed

    Ranea, Juan A G; Yeats, Corin; Grant, Alastair; Orengo, Christine A

    2007-11-01

    "Phylogenetic profiling" is based on the hypothesis that during evolution functionally or physically interacting genes are likely to be inherited or eliminated in a codependent manner. Creating presence-absence profiles of orthologous genes is now a common and powerful way of identifying functionally associated genes. In this approach, correctly determining orthology, as a means of identifying functional equivalence between two genes, is a critical and nontrivial step and largely explains why previous work in this area has mainly focused on using presence-absence profiles in prokaryotic species. Here, we demonstrate that eukaryotic genomes have a high proportion of multigene families whose phylogenetic profile distributions are poor in presence-absence information content. This feature makes them prone to orthology mis-assignment and unsuited to standard profile-based prediction methods. Using CATH structural domain assignments from the Gene3D database for 13 complete eukaryotic genomes, we have developed a novel modification of the phylogenetic profiling method that uses genome copy number of each domain superfamily to predict functional relationships. In our approach, superfamilies are subclustered at ten levels of sequence identity-from 30% to 100%-and phylogenetic profiles built at each level. All the profiles are compared using normalised Euclidean distances to identify those with correlated changes in their domain copy number. We demonstrate that two protein families will "auto-tune" with strong co-evolutionary signals when their profiles are compared at the similarity levels that capture their functional relationship. Our method finds functional relationships that are not detectable by the conventional presence-absence profile comparisons, and it does not require a priori any fixed criteria to define orthologous genes. PMID:18052542

  10. The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes

    PubMed Central

    Belkorchia, Abdel; Gasc, Cyrielle; Polonais, Valérie; Parisot, Nicolas; Gallois, Nicolas; Ribière, Céline; Lerat, Emmanuelle; Gaspin, Christine; Pombert, Jean-François; Peyret, Pierre; Peyretaillade, Eric

    2015-01-01

    The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms. PMID:26421846

  11. Evolutionary implications of intron-exon distribution and the properties and sequences of the RPL10A gene in eukaryotes.

    PubMed

    Del Campo, Eva M; Casano, Leonardo M; Barreno, Eva

    2013-03-01

    The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon-intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.

  12. Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.

    PubMed

    Hirst, Marissa B; Kita, Kelley N; Dawson, Scott C

    2011-01-01

    Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages

  13. Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.

    PubMed

    Hirst, Marissa B; Kita, Kelley N; Dawson, Scott C

    2011-01-01

    Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages

  14. Origins and evolution of viruses of eukaryotes: The ultimate modularity

    SciTech Connect

    Koonin, Eugene V.; Dolja, Valerian V.; Krupovic, Mart

    2015-05-15

    Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order “Megavirales” that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources

  15. Origins and evolution of viruses of eukaryotes: The ultimate modularity.

    PubMed

    Koonin, Eugene V; Dolja, Valerian V; Krupovic, Mart

    2015-05-01

    Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order "Megavirales" that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along

  16. Evolution of the RNA-dependent RNA polymerase (RdRP) genes: duplications and possible losses before and after the divergence of major eukaryotic groups.

    PubMed

    Zong, Jie; Yao, Xuan; Yin, Jinyuan; Zhang, Dabing; Ma, Hong

    2009-11-01

    Eukaryotic RNA-dependent RNA polymerases (RdRPs, encoded by RDR genes) play critical roles in developmental regulation, maintenance of genome integrity, and defense against foreign nucleic acids. However, the phylogenetic relationship of RDRs remains unclear. From available genome sequences, we identified 161 putative RDR genes from 56 eukaryotes, ranging from protists to multicellular organisms, including plants, fungi and invertebrate animals, such as nematodes, lancelet and sea anemone. On the other hand, we did not detect RDR homologs in vertebrates and insects, even though RNA interference functions in these organisms. Our phylogenetic analysis of the RDR genes suggests that the eukaryotic ancestor might have had three copies, i.e. RDRalpha, RDRbeta and RDRgamma. These three ancient copies were also supported by the patterns of protein sequence motifs. Further duplication events after the divergence of major eukaryotic groups were supported by the phylogenetic analyses, including some that likely occurred before the separation of subgroups within each kingdom. We present a model for a possible evolutionary history of RDR genes in eukaryotes.

  17. The tree of eukaryotes.

    PubMed

    Keeling, Patrick J; Burger, Gertraud; Durnford, Dion G; Lang, B Franz; Lee, Robert W; Pearlman, Ronald E; Roger, Andrew J; Gray, Michael W

    2005-12-01

    Recent advances in resolving the tree of eukaryotes are converging on a model composed of a few large hypothetical 'supergroups', each comprising a diversity of primarily microbial eukaryotes (protists, or protozoa and algae). The process of resolving the tree involves the synthesis of many kinds of data, including single-gene trees, multigene analyses, and other kinds of molecular and structural characters. Here, we review the recent progress in assembling the tree of eukaryotes, describing the major evidence for each supergroup, and where gaps in our knowledge remain. We also consider other factors emerging from phylogenetic analyses and comparative genomics, in particular lateral gene transfer, and whether such factors confound our understanding of the eukaryotic tree.

  18. Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia.

    PubMed

    Hugerth, Luisa W; Muller, Emilie E L; Hu, Yue O O; Lebrun, Laura A M; Roume, Hugo; Lundin, Daniel; Wilmes, Paul; Andersson, Anders F

    2014-01-01

    High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.

  19. Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.

    PubMed

    Vasiliou, V; Bairoch, A; Tipton, K F; Nebert, D W

    1999-08-01

    As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out

  20. PAT-seq: a method to study the integration of 3'-UTR dynamics with gene expression in the eukaryotic transcriptome.

    PubMed

    Harrison, Paul F; Powell, David R; Clancy, Jennifer L; Preiss, Thomas; Boag, Peter R; Traven, Ana; Seemann, Torsten; Beilharz, Traude H

    2015-08-01

    A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide. PMID:26092945

  1. PAT-seq: a method to study the integration of 3'-UTR dynamics with gene expression in the eukaryotic transcriptome.

    PubMed

    Harrison, Paul F; Powell, David R; Clancy, Jennifer L; Preiss, Thomas; Boag, Peter R; Traven, Ana; Seemann, Torsten; Beilharz, Traude H

    2015-08-01

    A major objective of systems biology is to quantitatively integrate multiple parameters from genome-wide measurements. To integrate gene expression with dynamics in poly(A) tail length and adenylation site, we developed a targeted next-generation sequencing approach, Poly(A)-Test RNA-sequencing. PAT-seq returns (i) digital gene expression, (ii) polyadenylation site/s, and (iii) the polyadenylation-state within and between eukaryotic transcriptomes. PAT-seq differs from previous 3' focused RNA-seq methods in that it depends strictly on 3' adenylation within total RNA samples and that the full-native poly(A) tail is included in the sequencing libraries. Here, total RNA samples from budding yeast cells were analyzed to identify the intersect between adenylation state and gene expression in response to loss of the major cytoplasmic deadenylase Ccr4. Furthermore, concordant changes to gene expression and adenylation-state were demonstrated in the classic Crabtree-Warburg metabolic shift. Because all polyadenylated RNA is interrogated by the approach, alternative adenylation sites, noncoding RNA and RNA-decay intermediates were also identified. Most important, the PAT-seq approach uses standard sequencing procedures, supports significant multiplexing, and thus replication and rigorous statistical analyses can for the first time be brought to the measure of 3'-UTR dynamics genome wide.

  2. The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cauliflower Or gene affects plant growth and development in addition to conferring beta-carotene accumulation. This study was undertaken to investigate the molecular basis of the Or gene mutation in controlling plant growth. The OR protein was found to interact with cauliflower and Arabidopsis e...

  3. Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Fan, Zhun; Chen, Jun; Wu, Jinxia; Li, Jiancheng

    2011-09-01

    The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii. PMID:21664493

  4. Group II Intron-Mediated Trans-Splicing in the Gene-Rich Mitochondrial Genome of an Enigmatic Eukaryote, Diphylleia rotans.

    PubMed

    Kamikawa, Ryoma; Shiratori, Takashi; Ishida, Ken-Ichiro; Miyashita, Hideaki; Roger, Andrew J

    2016-02-01

    Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans-splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans-splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia, which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit (cox) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans-splicing. This is the first example of group II intron-mediated trans-splicing outside Chloroplastida. Similar trans-splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans-splicing in D. rotans as well as in eukaryotes. PMID:26833505

  5. Group II Intron-Mediated Trans-Splicing in the Gene-Rich Mitochondrial Genome of an Enigmatic Eukaryote, Diphylleia rotans

    PubMed Central

    Kamikawa, Ryoma; Shiratori, Takashi; Ishida, Ken-Ichiro; Miyashita, Hideaki; Roger, Andrew J.

    2016-01-01

    Although mitochondria have evolved from a single endosymbiotic event, present day mitochondria of diverse eukaryotes display a great range of genome structures, content and features. Group I and group II introns are two features that are distributed broadly but patchily in mitochondrial genomes across branches of the tree of eukaryotes. While group I intron-mediated trans-splicing has been reported from some lineages distantly related to each other, findings of group II intron-mediated trans-splicing has been restricted to members of the Chloroplastida. In this study, we found the mitochondrial genome of the unicellular eukaryote Diphylleia rotans possesses currently the second largest gene repertoire. On the basis of a probable phylogenetic position of Diphylleia, which is located within Amorphea, current mosaic gene distribution in Amorphea must invoke parallel gene losses from mitochondrial genomes during evolution. Most notably, although the cytochrome c oxidase subunit (cox) 1 gene was split into four pieces which located at a distance to each other, we confirmed that a single mature mRNA that covered the entire coding region could be generated by group II intron-mediated trans-splicing. This is the first example of group II intron-mediated trans-splicing outside Chloroplastida. Similar trans-splicing mechanisms likely work for bipartitely split cox2 and nad3 genes to generate single mature mRNAs. We finally discuss origin and evolution of this type of trans-splicing in D. rotans as well as in eukaryotes. PMID:26833505

  6. Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector.

    PubMed

    Senejani, Alireza G; Sweasy, Joann B

    2010-01-01

    Escherichia coli (E. coli) are commonly used as hosts for DNA cloning and sequencing. Upon transformation of E. coli with recombined vector carrying a gene of interest, the bacteria multiply the gene of interest while maintaining the integrity of its content. During the subcloning of a mouse genomic fragment into a plasmid vector, we noticed that the size of the insert increased significantly upon replication in E. coli. The sequence of the insert was determined and found to contain a novel DNA sequence within the mouse genomic insert. A BLAST search of GenBank revealed the novel sequence to be that of the Insertion Sequence 2 (IS2) element from E. coli that was likely inserted during replication in that organism. Importantly, a detailed search of GenBank shows that the IS2 is present within many eukaryotic nucleotide sequences, and in many cases, has been annotated as being part of the protein. The results of this study suggest that one must perform additional careful analysis of the sequence results using BLAST comparisons, and further verification of gene annotation before submission into the GenBank. PMID:20678256

  7. Evolution of bacterial-like phosphoprotein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer.

    PubMed

    Uhrig, R Glen; Kerk, David; Moorhead, Greg B

    2013-12-01

    Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

  8. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    SciTech Connect

    Lee, A L

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  9. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    PubMed

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  10. Plastid 16S rRNA Gene Diversity among Eukaryotic Picophytoplankton Sorted by Flow Cytometry from the South Pacific Ocean

    PubMed Central

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J.; Vaulot, Daniel

    2011-01-01

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image. PMID:21552558

  11. Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

    PubMed Central

    Hoque, Mainul; Hanauske-Abel, Hartmut M; Palumbo, Paul; Saxena, Deepti; D'Alliessi Gandolfi, Darlene; Park, Myung Hee; Pe'ery, Tsafi; Mathews, Michael B

    2009-01-01

    Background Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. Results Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. Conclusion At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs. PMID:19825182

  12. Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.

    PubMed Central

    Hagiwara, K; Stenman, G; Honda, H; Sahlin, P; Andersson, A; Miyazono, K; Heldin, C H; Ishikawa, F; Takaku, F

    1991-01-01

    Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines. Images PMID:2005900

  13. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.

    PubMed Central

    Wu, J; Issa, J P; Herman, J; Bassett, D E; Nelkin, B D; Baylin, S B

    1993-01-01

    Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility. Images Fig. 1 Fig. 2 PMID:8415627

  14. Molecular phylogeny of eukaryotes.

    PubMed

    Schlegel, M

    1994-09-01

    Comparisons of ribosomal RNAs and various protein coding genes have contributed to a new view of eukaryote phylogeny. Analyses of paralogous protein coding genes suggest that archaebacteria and eukaryotes are sistergroups. Sequence diversity of small subunit rRNAs in protists by far exceeds that of any multicellular or prokaryote taxon. Remarkably, a group of taxa that lack mitochondria first branches off in the small subunit rRNA tree. The later radiations are formed by a series of clades that were once thought to be more ancestral. Furthermore, tracing of the evolutionary origin of secondary endobiontic events is now possible with sequence comparisons.

  15. Operons in eukaryotes.

    PubMed

    Blumenthal, Thomas

    2004-11-01

    It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

  16. Evidence that eukaryotic triosephosphate isomerase is of alpha-proteobacterial origin

    PubMed Central

    Keeling, Patrick J.; Doolittle, W. Ford

    1997-01-01

    We have cloned and sequenced genes for triosephosphate isomerase (TPI) from the gamma-proteobacterium Francisella tularensis, the green non-sulfur bacterium Chloroflexus aurantiacus, and the alpha-proteobacterium Rhizobium etli and used these in phylogenetic analysis with TPI sequences from other members of the Bacteria, Archaea, and Eukarya. These analyses show that eukaryotic TPI genes are most closely related to the homologue from the alpha-proteobacterium and most distantly related to archaebacterial homologues. This relationship suggests that the TPI genes present in modern eukaryotic genomes were derived from an alpha-proteobacterial genome (possibly that of the protomitochondrial endosymbiont) after the divergence of Archaea and Eukarya. Among these eukaryotic genes are some from deeply branching, amitochondrial eukaryotes (namely Giardia), which further suggests that this event took place quite early in eukaryotic evolution. PMID:9037042

  17. Homologous genes for mouse 4.5S hybRNA are found in all eukaryotes and their low molecular weight RNA transcripts intermolecularly hybridize with eukaryotic 18S ribosomal RNAs.

    PubMed

    Trinh-Rohlik, Q; Maxwell, E S

    1988-07-11

    Previous work has reported the isolation and sequencing of a mouse low molecular weight RNA species designated 4.5S hybridizing RNA or hybRNA because of its ability to intermolecularly hybridize with mouse mRNA and 18S rRNA sequences. Using synthetic DNA oligonucleotide probes we have examined the conservation of this gene sequence and its expression as a lmwRNA transcript across evolution. Southern blot analysis has shown that homologous genes of single or low copy number are found in all eukaryotes examined as well as in E. coli. Northern blot analysis has demonstrated 4.5S hybRNA transcription in all mouse tissues as well as expression in yeast and Xenopus laevis as lmwRNAs of approximately 130 and 100 nucleotides, respectively, as compared with mouse/rat/hamster species of approximately 87 nucleotides. Yeast and X. laevis 4.5S hybRNA homologs, isolated by hybrid-selection, were shown by Northern blot analysis to intermolecularly hybridize with homologous as well as heterologous 18S rRNA sequences. The conservation of 4.5S hybRNA homologous genes and their expression as lmwRNA transcripts with common intermolecular RNA:RNA hybridization capabilities in fungi, amphibians, and mammals argues for a common, conserved and required biological function for this lmwRNA in all eukaryotes and potential utilization of its intermolecular RNA:RNA hybridization capabilities to carry out this function.

  18. Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

    PubMed

    Nagano, Naoki; Sakaguchi, Keishi; Taoka, Yousuke; Okita, Yuji; Honda, Daiske; Ito, Makoto; Hayashi, Masahiro

    2011-01-01

    Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms.

  19. Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

    PubMed

    Díez, B; Pedrós-Alió, C; Massana, R

    2001-07-01

    Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

  20. Evolution of early eukaryotic cells: genomes, proteomes, and compartments.

    PubMed

    Bogorad, Lawrence

    2008-01-01

    Eukaryotes arose from an endosymbiotic association of an alpha-proteobacterium-like organism (the ancestor of mitochondria) with a host cell (lacking mitochondria or plastids). Plants arose by the addition of a cyanobacterium-like endosymbiont (the ancestor of plastids) to the two-member association. Each member of the association brought a unique internal environment and a unique genome. Analyses of recently acquired genomic sequences with newly developed algorithms have revealed (a) that the number of endosymbiont genes that remain in eukaryotic cells-principally in the nucleus-is surprisingly large, (b) that protein products of a large number of genes (or their descendents) that entered the association in the genome of the host are now directed to an organelle derived from an endosymbiont, and (c) that protein products of genes traceable to endosymbiont genomes are directed to the nucleo-cytoplasmic compartment. Consideration of these remarkable findings has led to the present suggestion that contemporary eukaryotic cells evolved through continual chance relocation and testing of genes as well as combinations of gene products and biochemical processes in each unique cell compartment derived from a member of the eukaryotic association. Most of these events occurred during about 300 million years, or so, before contemporary forms of eukaryotic cells appear in the fossil record; they continue today. PMID:17912611

  1. Eukaryotic origins.

    PubMed

    Lake, James A

    2015-09-26

    The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques.

  2. Eukaryotic origins

    PubMed Central

    Lake, James A.

    2015-01-01

    The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques. PMID:26323753

  3. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif

    PubMed Central

    Bürglin, Thomas R

    2008-01-01

    Background The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini. Results We performed extensive genome searches such as the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria. Conclusion In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are found in many protists indicates that this gene family must have

  4. The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids.

    PubMed

    Nozaki, Hisayoshi; Matsuzaki, Motomichi; Takahara, Manabu; Misumi, Osami; Kuroiwa, Haruko; Hasegawa, Masami; Shin-i, Tadasu; Kohara, Yuji; Ogasawara, Naotake; Kuroiwa, Tsuneyoshi

    2003-04-01

    Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (alpha- and beta-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1alpha, alpha-tubulin, and beta-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various

  5. Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

    PubMed

    Huys, Geert; Vanhoutte, Tom; Joossens, Marie; Mahious, Amal S; De Brandt, Evie; Vermeire, Severine; Swings, Jean

    2008-06-01

    The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.

  6. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    PubMed

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  7. Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18S rRNA genes.

    PubMed

    Lie, Alle A Y; Liu, Zhenfeng; Hu, Sarah K; Jones, Adriane C; Kim, Diane Y; Countway, Peter D; Amaral-Zettler, Linda A; Cary, S Craig; Sherr, Evelyn B; Sherr, Barry F; Gast, Rebecca J; Caron, David A

    2014-07-01

    Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

  8. Gene expression as a circular process: cross-talk between transcription and mRNA degradation in eukaryotes; International University of Andalusia (UNIA) Baeza, Spain.

    PubMed

    Collart, Martine A; Reese, Joseph C

    2014-01-01

    Studies on the regulation of gene expression in eukaryotes in the past 20 years have consistently revealed increasing levels of complexity. Thirty years ago it seemed that we had understood the basic principles of gene regulation in eukaryotes. It was thought that regulation of transcription was the first and most important stage at which gene expression was regulated, and transcriptional regulation was considered to be very simple, with DNA-binding activators and repressors talking to the basic transcription machinery. This simple model was overthrown when it became clear that other stages of gene expression are also highly regulated. More recently, other dogmas have started to collapse. In particular, the idea that a linkage between the different steps in gene expression is restricted to processes ongoing in the same compartment has fallen out of favor. It is now evident that functional and physical linkage occurs in eukaryotes. We know that factors contributing to transcription in the nucleus can be found in the cytoplasm, and that RNA binding proteins that contribute to RNA decay in the cytoplasm are present in the nucleus. However, shuttling of such factors between nucleus and cytoplasm has traditionally been thought to serve a simple regulatory purpose, for instance, to avoid untimely activation of a transcription factor in the nucleus. Alternatively, it was thought to be necessary to recruit RNA binding proteins to the relevant RNAs. The notion that is now emerging is that factors thought to have evolved to specialize in regulating a single step of gene regulation in one cellular compartment may be contributing to the regulation of mRNAs at multiple steps along the lifecycle of an mRNA.

  9. Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus

    PubMed Central

    Romano, Stefano; Fernàndez-Guerra, Antonio; Reen, F. Jerry; Glöckner, Frank O.; Crowley, Susan P.; O'Sullivan, Orla; Cotter, Paul D.; Adams, Claire; Dobson, Alan D. W.; O'Gara, Fergal

    2016-01-01

    Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its

  10. Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus.

    PubMed

    Romano, Stefano; Fernàndez-Guerra, Antonio; Reen, F Jerry; Glöckner, Frank O; Crowley, Susan P; O'Sullivan, Orla; Cotter, Paul D; Adams, Claire; Dobson, Alan D W; O'Gara, Fergal

    2016-01-01

    Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its

  11. PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

    PubMed

    Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

    2015-11-01

    Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

  12. Prokaryote and eukaryote evolvability.

    PubMed

    Poole, Anthony M; Phillips, Matthew J; Penny, David

    2003-05-01

    The concept of evolvability covers a broad spectrum of, often contradictory, ideas. At one end of the spectrum it is equivalent to the statement that evolution is possible, at the other end are untestable post hoc explanations, such as the suggestion that current evolutionary theory cannot explain the evolution of evolvability. We examine similarities and differences in eukaryote and prokaryote evolvability, and look for explanations that are compatible with a wide range of observations. Differences in genome organisation between eukaryotes and prokaryotes meets this criterion. The single origin of replication in prokaryote chromosomes (versus multiple origins in eukaryotes) accounts for many differences because the time to replicate a prokaryote genome limits its size (and the accumulation of junk DNA). Both prokaryotes and eukaryotes appear to switch from genetic stability to genetic change in response to stress. We examine a range of stress responses, and discuss how these impact on evolvability, particularly in unicellular organisms versus complex multicellular ones. Evolvability is also limited by environmental interactions (including competition) and we describe a model that places limits on potential evolvability. Examples are given of its application to predator competition and limits to lateral gene transfer. We suggest that unicellular organisms evolve largely through a process of metabolic change, resulting in biochemical diversity. Multicellular organisms evolve largely through morphological changes, not through extensive changes to cellular biochemistry. PMID:12689728

  13. Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

    PubMed Central

    Mori, Hiroshi; Maruyama, Fumito; Kato, Hiromi; Toyoda, Atsushi; Dozono, Ayumi; Ohtsubo, Yoshiyuki; Nagata, Yuji; Fujiyama, Asao; Tsuda, Masataka; Kurokawa, Ken

    2014-01-01

    The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities. PMID:24277737

  14. Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes.

    PubMed

    Mori, Hiroshi; Maruyama, Fumito; Kato, Hiromi; Toyoda, Atsushi; Dozono, Ayumi; Ohtsubo, Yoshiyuki; Nagata, Yuji; Fujiyama, Asao; Tsuda, Masataka; Kurokawa, Ken

    2014-01-01

    The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.

  15. Symbiosis and the origin of eukaryotic motility

    NASA Technical Reports Server (NTRS)

    Margulis, L.; Hinkle, G.

    1991-01-01

    Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

  16. Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

    PubMed Central

    Stratikos, E.; Alberdi, E.; Gettins, P. G.; Becerra, S. P.

    1996-01-01

    Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation. PMID:8976566

  17. Biology wars: the eukaryotes strike back.

    PubMed

    Dunning Hotopp, Julie C; Estes, Anne M

    2014-12-10

    It is increasingly clear that eukaryotes have acquired bacterial DNA and function through horizontal gene transfer (HGT). In this issue of Cell Host & Microbe, Chou et al. (2014) and Metcalf et al. (2014) report multiple HGTs of bacterial tae and lysozyme genes, respectively, to diverse eukaryotic and archaeal hosts that may complement their response to bacteria.

  18. Bacteriophage-Derived Vectors for Targeted Cancer Gene Therapy

    PubMed Central

    Pranjol, Md Zahidul Islam; Hajitou, Amin

    2015-01-01

    Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration. PMID:25606974

  19. Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes.

    PubMed

    Nakashima, H; Grahovac, M J; Mazzarella, R; Fujiwara, H; Kitchen, J R; Threat, T A; Ko, M S

    1999-09-01

    Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.

  20. Evolutionary appearance of genes encoding proteins associated with box H/ACA snoRNAs: Cbf5p in Euglena gracilis, an early diverging eukaryote, and candidate Gar1p and Nop10p homologs in archaebacteria

    PubMed Central

    Watanabe, Yoh-ichi; Gray, Michael W.

    2000-01-01

    A reverse transcription–polymerase chain reaction (RT–PCR) approach was used to clone a cDNA encoding the Euglena gracilis homolog of yeast Cbf5p, a protein component of the box H/ACA class of snoRNPs that mediate pseudouridine formation in eukaryotic rRNA. Cbf5p is a putative pseudouridine synthase, and the Euglena homolog is the first full-length Cbf5p sequence to be reported for an early diverging unicellular eukaryote (protist). Phylogenetic analysis of putative pseudouridine synthase sequences confirms that archaebacterial and eukaryotic (including Euglena) Cbf5p proteins are specifically related and are distinct from the TruB/Pus4p clade that is responsible for formation of pseudouridine at position 55 in eubacterial (TruB) and eukaryotic (Pus4p) tRNAs. Using a bioinformatics approach, we also identified archaebacterial genes encoding candidate homologs of yeast Gar1p and Nop10p, two additional proteins known to be associated with eukaryotic box H/ACA snoRNPs. These observations raise the possibility that pseudouridine formation in archaebacterial rRNA may be dependent on analogs of the eukaryotic box H/ACA snoRNPs, whose evolutionary origin may therefore predate the split between Archaea (archaebacteria) and Eucarya (eukaryotes). Database searches further revealed, in archaebacterial and some eukaryotic genomes, two previously unrecognized groups of genes (here designated ‘PsuX’ and ‘PsuY’) distantly related to the Cbf5p/TruB gene family. PMID:10871366

  1. Plastid-derived genes in the nonphotosynthetic alveolate Oxyrrhis marina.

    PubMed

    Slamovits, Claudio H; Keeling, Patrick J

    2008-07-01

    Reconstructing the history of plastid acquisition and loss in the alveolate protists is a difficult problem because our knowledge of the distribution of plastids in extant lineages is incomplete due to the possible presence of cryptic, nonphotosynthetic plastids in several lineages. The discovery of the apicoplast in apicomplexan parasites has drawn attention to this problem and, more specifically, to the question of whether many other nonphotosynthetic lineages also contain cryptic plastids or are derived from plastid-containing ancestors. Oxyrrhis marina is one such organism: It is a heterotrophic, early-branching member of the dinoflagellate lineage for which there is no evidence of a plastid. To investigate the possibility that O. marina is derived from a photosynthetic ancestor, we have generated and analyzed a large-scale EST database and searched for evidence of plastid-derived genes. Here, we describe 8 genes whose phylogeny shows them to be derived from plastid-targeted homologues. These genes encode proteins from several pathways known to be localized in the plastids of other algae, including synthesis of tetrapyrroles, isoprenoids, and amino acids, as well as carbon metabolism and oxygen detoxification. The 5' end of 5 cDNAs were also characterized using cap-dependent or spliced leader-mediated reverse transcriptase-polymerase chain reaction, revealing that at least 4 of these genes have retained leaders that are similar in nature to the plastid-targeting signals of other secondary plastids, suggesting that these proteins may be targeted to a cryptic organelle. At least 2 genes do not encode such leaders, and their products may presently function in the cytosol. Altogether, the presence of plastid-derived genes in O. marina shows that its ancestors contained a plastid, and the pathways represented by the genes and presence of targeting signals on at least some of the genes further suggests that a relict organelle may still exist to fulfill plastid

  2. A New Class of SINEs with snRNA Gene-Derived Heads.

    PubMed

    Kojima, Kenji K

    2015-05-27

    Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5' region (head) of the majority of SINEs is derived from one of the three types of RNA genes--7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)--and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5' end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar.

  3. A New Class of SINEs with snRNA Gene-Derived Heads.

    PubMed

    Kojima, Kenji K

    2015-06-01

    Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5' region (head) of the majority of SINEs is derived from one of the three types of RNA genes--7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)--and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5' end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar. PMID:26019167

  4. What was the real contribution of endosymbionts to the eukaryotic nucleus? Insights from photosynthetic eukaryotes.

    PubMed

    Moreira, David; Deschamps, Philippe

    2014-07-01

    Eukaryotic genomes are composed of genes of different evolutionary origins. This is especially true in the case of photosynthetic eukaryotes, which, in addition to typical eukaryotic genes and genes of mitochondrial origin, also contain genes coming from the primary plastids and, in the case of secondary photosynthetic eukaryotes, many genes provided by the nuclei of red or green algal endosymbionts. Phylogenomic analyses have been applied to detect those genes and, in some cases, have led to proposing the existence of cryptic, no longer visible endosymbionts. However, detecting them is a very difficult task because, most often, those genes were acquired a long time ago and their phylogenetic signal has been heavily erased. We revisit here two examples, the putative cryptic endosymbiosis of green algae in diatoms and chromerids and of Chlamydiae in the first photosynthetic eukaryotes. We show that the evidence sustaining them has been largely overestimated, and we insist on the necessity of careful, accurate phylogenetic analyses to obtain reliable results.

  5. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5' UTRs and its implications for eukaryotic gene translation regulation.

    PubMed

    Pánek, Josef; Kolár, Michal; Vohradský, Jirí; Shivaya Valásek, Leos

    2013-09-01

    There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA-rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5' untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5' UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5' UTRs of mRNAs.

  6. Arabinogalactan proteins have deep roots in eukaryotes: identification of genes and epitopes in brown algae and their role in Fucus serratus embryo development.

    PubMed

    Hervé, Cécile; Siméon, Amandine; Jam, Murielle; Cassin, Andrew; Johnson, Kim L; Salmeán, Armando A; Willats, William G T; Doblin, Monika S; Bacic, Antony; Kloareg, Bernard

    2016-03-01

    Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which is unrelated to land plants and green algae (Chloroplastida). Brown algae share common evolutionary features with other multicellular organisms, including a carbohydrate-rich cell wall. They differ markedly from plants in their cell wall composition, and AGPs have not been reported in brown algae. Here we investigated the presence of chimeric AGP-like core proteins in this lineage. We report that the genome sequence of the brown algal model Ectocarpus siliculosus encodes AGP protein backbone motifs, in a gene context that differs considerably from what is known in land plants. We showed the occurrence of AGP glycan epitopes in a range of brown algal cell wall extracts. We demonstrated that these chimeric AGP-like core proteins are developmentally regulated in embryos of the order Fucales and showed that AGP loss of function seriously impairs the course of early embryogenesis. Our findings shine a new light on the role of AGPs in cell wall sensing and raise questions about the origin and evolution of AGPs in eukaryotes.

  7. Arabinogalactan proteins have deep roots in eukaryotes: identification of genes and epitopes in brown algae and their role in Fucus serratus embryo development.

    PubMed

    Hervé, Cécile; Siméon, Amandine; Jam, Murielle; Cassin, Andrew; Johnson, Kim L; Salmeán, Armando A; Willats, William G T; Doblin, Monika S; Bacic, Antony; Kloareg, Bernard

    2016-03-01

    Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which is unrelated to land plants and green algae (Chloroplastida). Brown algae share common evolutionary features with other multicellular organisms, including a carbohydrate-rich cell wall. They differ markedly from plants in their cell wall composition, and AGPs have not been reported in brown algae. Here we investigated the presence of chimeric AGP-like core proteins in this lineage. We report that the genome sequence of the brown algal model Ectocarpus siliculosus encodes AGP protein backbone motifs, in a gene context that differs considerably from what is known in land plants. We showed the occurrence of AGP glycan epitopes in a range of brown algal cell wall extracts. We demonstrated that these chimeric AGP-like core proteins are developmentally regulated in embryos of the order Fucales and showed that AGP loss of function seriously impairs the course of early embryogenesis. Our findings shine a new light on the role of AGPs in cell wall sensing and raise questions about the origin and evolution of AGPs in eukaryotes. PMID:26667994

  8. Eukaryotic evolution: getting to the root of the problem.

    PubMed

    Simpson, Alastair G B; Roger, Andrew J

    2002-10-15

    Comparative analyses of multiple genes suggest most known eukaryotes can be classified into half a dozen 'super-groups'. A new investigation of the distribution of a fused gene pair amongst these 'super-groups' has greatly narrowed the possible positions of the root of the eukaryote tree, clarifying the broad outlines of early eukaryote evolution.

  9. Solution structure of subunit F (Vma7p) of the eukaryotic V(1)V(O) ATPase from Saccharomyces cerevisiae derived from SAXS and NMR spectroscopy.

    PubMed

    Basak, Sandip; Gayen, Shovanlal; Thaker, Youg R; Manimekalai, Malathy S S; Roessle, Manfred; Hunke, Cornelia; Grüber, Gerhard

    2011-01-01

    Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A(3)B(3) sector of the V(1) ATPase to pump protons via the membrane-embedded V(O) sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F(1-94) was produced and analyzed by SAXS. Comparison of the F(1-94) shape with the one of subunit F showed the missing hook-like region in F(1-94), supported by the decreased D(max) value of F(1-94) (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F(90-116), was solved, displaying an α-helical region between residues 103 and 113. The F(90-116) solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V(1) ATPase is discussed. PMID:20840841

  10. An RNA-dependent RNA polymerase gene in bat genomes derived from an ancient negative-strand RNA virus.

    PubMed

    Horie, Masayuki; Kobayashi, Yuki; Honda, Tomoyuki; Fujino, Kan; Akasaka, Takumi; Kohl, Claudia; Wibbelt, Gudrun; Mühldorfer, Kristin; Kurth, Andreas; Müller, Marcel A; Corman, Victor M; Gillich, Nadine; Suzuki, Yoshiyuki; Schwemmle, Martin; Tomonaga, Keizo

    2016-01-01

    Endogenous bornavirus-like L (EBLL) elements are inheritable sequences derived from ancient bornavirus L genes that encode a viral RNA-dependent RNA polymerase (RdRp) in many eukaryotic genomes. Here, we demonstrate that bats of the genus Eptesicus have preserved for more than 11.8 million years an EBLL element named eEBLL-1, which has an intact open reading frame of 1,718 codons. The eEBLL-1 coding sequence revealed that functional motifs essential for mononegaviral RdRp activity are well conserved in the EBLL-1 genes. Genetic analyses showed that natural selection operated on eEBLL-1 during the evolution of Eptesicus. Notably, we detected efficient transcription of eEBLL-1 in tissues from Eptesicus bats. To the best of our knowledge, this study is the first report showing that the eukaryotic genome has gained a riboviral polymerase gene from an ancient virus that has the potential to encode a functional RdRp. PMID:27174689

  11. An RNA-dependent RNA polymerase gene in bat genomes derived from an ancient negative-strand RNA virus

    PubMed Central

    Horie, Masayuki; Kobayashi, Yuki; Honda, Tomoyuki; Fujino, Kan; Akasaka, Takumi; Kohl, Claudia; Wibbelt, Gudrun; Mühldorfer, Kristin; Kurth, Andreas; Müller, Marcel A.; Corman, Victor M.; Gillich, Nadine; Suzuki, Yoshiyuki; Schwemmle, Martin; Tomonaga, Keizo

    2016-01-01

    Endogenous bornavirus-like L (EBLL) elements are inheritable sequences derived from ancient bornavirus L genes that encode a viral RNA-dependent RNA polymerase (RdRp) in many eukaryotic genomes. Here, we demonstrate that bats of the genus Eptesicus have preserved for more than 11.8 million years an EBLL element named eEBLL-1, which has an intact open reading frame of 1,718 codons. The eEBLL-1 coding sequence revealed that functional motifs essential for mononegaviral RdRp activity are well conserved in the EBLL-1 genes. Genetic analyses showed that natural selection operated on eEBLL-1 during the evolution of Eptesicus. Notably, we detected efficient transcription of eEBLL-1 in tissues from Eptesicus bats. To the best of our knowledge, this study is the first report showing that the eukaryotic genome has gained a riboviral polymerase gene from an ancient virus that has the potential to encode a functional RdRp. PMID:27174689

  12. Comparative analysis of eukaryotic marine microbial assemblages from 18S rRNA gene and gene transcript clone libraries by using different methods of extraction.

    PubMed

    Koid, Amy; Nelson, William C; Mraz, Amy; Heidelberg, Karla B

    2012-06-01

    Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned.

  13. Homologs of mitochondrial transcription factor B, sparsely distributed within the eukaryotic radiation, are likely derived from the dimethyladenosine methyltransferase of the mitochondrial endosymbiont.

    PubMed

    Shutt, Timothy E; Gray, Michael W

    2006-06-01

    Mitochondrial transcription factor B (mtTFB), an essential component in regulating the expression of mitochondrial DNA-encoded genes in both yeast and humans, is a dimethyladenosine methyltransferase (DMT) that has acquired a secondary role in mitochondrial transcription. So far, mtTFB has only been well studied in Opisthokonta (metazoan animals and fungi). Here we investigate the phylogenetic distribution of mtTFB homologs throughout the domain Eucarya, documenting the first examples of this protein outside of the opisthokonts. Surprisingly, we identified putative mtTFB homologs only in amoebozoan protists and trypanosomatids. Phylogenetic analysis together with conservation of intron positions in amoebozoan and human genes supports the grouping of the putative mtTFB homologs as a distinct clade. Phylogenetic analysis further demonstrates that the mtTFB is most likely derived from the DMT of the mitochondrial endosymbiont.

  14. The relative ages of eukaryotes and akaryotes.

    PubMed

    Penny, David; Collins, Lesley J; Daly, Toni K; Cox, Simon J

    2014-12-01

    The Last Eukaryote Common Ancestor (LECA) appears to have the genetics required for meiosis, mitosis, nucleus and nuclear substructures, an exon/intron gene structure, spliceosomes, many centres of DNA replication, etc. (and including mitochondria). Most of these features are not generally explained by models for the origin of the Eukaryotic cell based on the fusion of an Archeon and a Bacterium. We find that the term 'prokaryote' is ambiguous and the non-phylogenetic term akaryote should be used in its place because we do not yet know the direction of evolution between eukaryotes and akaryotes. We use the term 'protoeukaryote' for the hypothetical stem group ancestral eukaryote that took up a bacterium as an endosymbiont that formed the mitochondrion. It is easier to make detailed models with a eukaryote to an akaryote transition, rather than vice versa. So we really are at a phylogenetic impasse in not being confident about the direction of change between eukaryotes and akaryotes.

  15. A Eukaryote without a Mitochondrial Organelle.

    PubMed

    Karnkowska, Anna; Vacek, Vojtěch; Zubáčová, Zuzana; Treitli, Sebastian C; Petrželková, Romana; Eme, Laura; Novák, Lukáš; Žárský, Vojtěch; Barlow, Lael D; Herman, Emily K; Soukal, Petr; Hroudová, Miluše; Doležal, Pavel; Stairs, Courtney W; Roger, Andrew J; Eliáš, Marek; Dacks, Joel B; Vlček, Čestmír; Hampl, Vladimír

    2016-05-23

    The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

  16. A Eukaryote without a Mitochondrial Organelle.

    PubMed

    Karnkowska, Anna; Vacek, Vojtěch; Zubáčová, Zuzana; Treitli, Sebastian C; Petrželková, Romana; Eme, Laura; Novák, Lukáš; Žárský, Vojtěch; Barlow, Lael D; Herman, Emily K; Soukal, Petr; Hroudová, Miluše; Doležal, Pavel; Stairs, Courtney W; Roger, Andrew J; Eliáš, Marek; Dacks, Joel B; Vlček, Čestmír; Hampl, Vladimír

    2016-05-23

    The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell. PMID:27185558

  17. Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways.

    PubMed

    Smirnova, Julia B; Selley, Julian N; Sanchez-Cabo, Fatima; Carroll, Kathleen; Eddy, A Alan; McCarthy, John E G; Hubbard, Simon J; Pavitt, Graham D; Grant, Chris M; Ashe, Mark P

    2005-11-01

    Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.

  18. Open Questions on the Origin of Eukaryotes.

    PubMed

    López-García, Purificación; Moreira, David

    2015-11-01

    Despite recent progress, the origin of the eukaryotic cell remains enigmatic. It is now known that the last eukaryotic common ancestor was complex and that endosymbiosis played a crucial role in eukaryogenesis at least via the acquisition of the alphaproteobacterial ancestor of mitochondria. However, the nature of the mitochondrial host is controversial, although the recent discovery of an archaeal lineage phylogenetically close to eukaryotes reinforces models proposing archaea-derived hosts. We argue that, in addition to improved phylogenomic analyses with more comprehensive taxon sampling to pinpoint the closest prokaryotic relatives of eukaryotes, determining plausible mechanisms and selective forces at the origin of key eukaryotic features, such as the nucleus or the bacterial-like eukaryotic membrane system, is essential to constrain existing models.

  19. Open questions on the origin of eukaryotes

    PubMed Central

    López-García, Purificación; Moreira, David

    2015-01-01

    Despite recent progress, the origin of the eukaryotic cell remains enigmatic. It is now known that the last eukaryotic common ancestor was complex and that endosymbiosis played a crucial role in eukaryogenesis at least via the acquisition of the alphaproteobacterial ancestor of mitochondria. However, the nature of the mitochondrial host is controversial, although the recent discovery of an archaeal lineage phylogenetically close to eukaryotes reinforces models proposing archaea-derived hosts. We argue that, in addition to improved phylogenomic analyses with more comprehensive taxon sampling to pinpoint the closest prokaryotic relatives of eukaryotes, determining plausible mechanisms and selective forces at the origin of key eukaryotic features, such as the nucleus or the bacterial-like eukaryotic membrane system, is essential to constrain existing models. PMID:26455774

  20. A New Class of SINEs with snRNA Gene-Derived Heads

    PubMed Central

    Kojima, Kenji K.

    2015-01-01

    Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5′ region (head) of the majority of SINEs is derived from one of the three types of RNA genes—7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)—and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5′ end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar. PMID:26019167

  1. Killing of cancer cells through the use of eukaryotic expression vectors harbouring genes encoding nucleases and ribonuclease inhibitor.

    PubMed

    Glinka, Elena M

    2015-05-01

    Cancer gene therapy vectors are promising tools for killing cancer cells with the purpose of eradicating malignant tumours entirely. Different delivery methods of vectors into the cancer cells, including both non-viral and viral, as well as promoters for the targeted expression of genes encoding anticancer proteins were developed for effective and selective killing of cancer cells without harming healthy cells. Many vectors have been created to kill cancer cells, and some vectors suppress malignant tumours with high efficiency. This review is focused on vectors bearing genes for nucleases such as deoxyribonucleases (caspase-activated DNase, deoxyribonuclease I-like 3, endonuclease G) and ribonucleases (human polynucleotide phosphorylase, ribonuclease L, α-sarcin, barnase), as well as vectors harbouring gene encoding ribonuclease inhibitor. The data concerning the functionality and the efficacy of such vectors are presented.

  2. Gene expression changes induced by Trypanosoma cruzi shed microvesicles in mammalian host cells: relevance of tRNA-derived halves.

    PubMed

    Garcia-Silva, Maria R; Cabrera-Cabrera, Florencia; das Neves, Roberta Ferreira Cura; Souto-Padrón, Thaís; de Souza, Wanderley; Cayota, Alfonso

    2014-01-01

    At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

  3. Handling tRNA introns, archaeal way and eukaryotic way

    PubMed Central

    Yoshihisa, Tohru

    2014-01-01

    Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3′ to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages. PMID:25071838

  4. Cloning and characterisation of the pknD gene encoding an eukaryotic-type protein kinase in the cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Zhang, C C; Libs, L

    1998-04-01

    Protein phosphorylation catalysed by protein kinases is an important mechanism for signal transduction in both prokaryotes and eukaryotes. A novel gene, pknD, encoding a protein similar to eukaryotic-type protein kinases, was cloned from Anabaena sp. PCC7120. The N-terminal region of PknD is 60% identical to that of PknA, another putative Ser/Thr kinase from the same strain. Both PknA and PknD have C-terminal regions that are rich in Pro and Thr residues. Expression of pknD was undetectable by RNA/DNA hybridisation and was thus examined by RT-PCR. The pknD transcript was detected in filaments cultured in the presence of either nitrate or ammonium as a source of combined nitrogen, and also in filaments transferred from nitrate-sufficient to N2-fixing conditions. pknD mutants were created, and their growth characteristics under different nitrogen regimes and their capacity for heterocyst development were investigated. The growth rates of the mutants were similar to those of the wild-type strain in the presence of either nitrate or ammonium, but were only 20% that of the wild type under N2-fixing conditions. The rate of nitrogenase activity is normal in pknD mutant under aerobic conditions. Under nitrogen-fixing conditions, the inactivation of pknD led to enhanced modification of the PII protein compared to the weak phosphorylation of PII observed in the wild-type strain. This high level of PII phosphorylation in the pknD mutant is reminiscent of the situation in nitrogen-starved Synechococcus PCC7942 cells. PknD might be involved in regulating nitrogen metabolism or nitrogen trafficking from heterocysts to vegetative cells.

  5. CpLEA5, the Late Embryogenesis Abundant Protein Gene from Chimonanthus praecox, Possesses Low Temperature and Osmotic Resistances in Prokaryote and Eukaryotes

    PubMed Central

    Liu, Yiling; Xie, Lixia; Liang, Xilong; Zhang, Shihong

    2015-01-01

    Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs) in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031). Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance. PMID:26569231

  6. [Structural-functional characteristics of the Schizosaccharomyces pombe rpb8+ gene, coding the subunit of RNA polymerase I-III, specific only for eukaryotes].

    PubMed

    Shpakovskiĭ, G V; Proshkin, S A; Kaiushin, A L; Korosteleva, M D; Lebedenko, E N

    1998-02-01

    A full-length cDNA of the rpb8+ gene encoding a common subunit Rpb8 of nuclear RNA polymerases I-III only specific for Eucarya was isolated from an expression library of the fission yeast Schizosaccharomyces pombe. The primary structure of the corresponding fragment of the Sz. pombe genome was also established. The rpb8+ gene contains two short introns, 59 and 48 bp long. Only short segments of homology were found upon comparing the Rpb8 subunit homologs from various eukaryotic species, and substantial differences exist between the corresponding proteins of unicellular and multicellular organisms. Subunit Rpb8 of Sz. pombe proved to be the smallest one among the known related proteins: it lacks the 21-aa fragment corresponding to amino acids residues 68-88 of the central part of the homologous subunit ABC14.5 of Saccharomyces cerevisiae. Accordingly, subunit Rpb8 of the fission yeast was not capable of substituting in vivo subunit ABC14.5 in nuclear RNA polymerases of the baker's yeast. PMID:10335407

  7. CpLEA5, the Late Embryogenesis Abundant Protein Gene from Chimonanthus praecox, Possesses Low Temperature and Osmotic Resistances in Prokaryote and Eukaryotes.

    PubMed

    Liu, Yiling; Xie, Lixia; Liang, Xilong; Zhang, Shihong

    2015-01-01

    Plants synthesize and accumulate a series of stress-resistance proteins to protect normal physiological activities under adverse conditions. Chimonanthus praecox which blooms in freezing weather accumulates late embryogenesis abundant proteins (LEAs) in flowers, but C. praecox LEAs are little reported. Here, we report a group of five LEA genes of C. praecox (CpLEA5, KT727031). Prokaryotic-expressed CpLEA5 was employed in Escherichia coli to investigate bioactivities and membrane permeability at low-temperature. In comparison with the vacant strains, CpLEA5-containing strains survived in a 20% higher rate; and the degree of cell membrane damage in CpLEA5-containing strains was 55% of that of the vacant strains according to a conductivity test, revealing the low-temperature resistance of CpLEA5 in bacteria. CpLEA5 was also expressed in Pichia pastoris. Interestingly, besides low-temperature resistance, CpLEA5 conferred high resistance to salt and alkali in CpLEA5 overexpressing yeast. The CpLEA5 gene was transferred into Arabidopsis thaliana to also demonstrate CpLEA5 actions in plants. As expected, the transgenic lines were more resistant against low-temperature and drought while compared with the wild type. Taken together, CpLEA5-conferred resistances to several conditions in prokaryote and eukaryotes could have great value as a genetic technology to enhance osmotic stress and low-temperature tolerance. PMID:26569231

  8. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    PubMed Central

    Shang, Jin; Fan, Xin; Shangguan, Lei; Liu, Huan; Zhou, Yue

    2015-01-01

    Low back pain (LBP) is a very prevalent disease and degenerative disc diseases (DDDs) usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0) to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale. PMID:26649308

  9. Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

    PubMed Central

    Schwan, W R; Kopecko, D J

    1997-01-01

    A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive. PMID:9353061

  10. A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E).

    PubMed

    Ruffel, Sandrine; Dussault, Marie-Hélène; Palloix, Alain; Moury, Benoît; Bendahmane, Abdelhafid; Robaglia, Christophe; Caranta, Carole

    2002-12-01

    We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype. PMID:12492847

  11. Heuristic approach to deriving models for gene finding.

    PubMed

    Besemer, J; Borodovsky, M

    1999-10-01

    Computer methods of accurate gene finding in DNA sequences require models of protein coding and non-coding regions derived either from experimentally validated training sets or from large amounts of anonymous DNA sequence. Here we propose a new, heuristic method producing fairly accurate inhomogeneous Markov models of protein coding regions. The new method needs such a small amount of DNA sequence data that the model can be built 'on the fly' by a web server for any DNA sequence >400 nt. Tests on 10 complete bacterial genomes performed with the GeneMark.hmm program demonstrated the ability of the new models to detect 93.1% of annotated genes on average, while models built by traditional training predict an average of 93.9% of genes. Models built by the heuristic approach could be used to find genes in small fragments of anonymous prokaryotic genomes and in genomes of organelles, viruses, phages and plasmids, as well as in highly inhomogeneous genomes where adjustment of models to local DNA composition is needed. The heuristic method also gives an insight into the mechanism of codon usage pattern evolution.

  12. Unique marine derived cyanobacterial biosynthetic genes for chemical diversity.

    PubMed

    Kleigrewe, Karin; Gerwick, Lena; Sherman, David H; Gerwick, William H

    2016-02-01

    Cyanobacteria are a prolific source of structurally unique and biologically active natural products that derive from intriguing biochemical pathways. Advancements in genome sequencing have accelerated the identification of unique modular biosynthetic gene clusters in cyanobacteria and reveal a wealth of unusual enzymatic reactions involved in their construction. This article examines several interesting mechanistic transformations involved in cyanobacterial secondary metabolite biosynthesis with a particular focus on marine derived modular polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS) and combinations thereof to form hybrid natural products. Further, we focus on the cyanobacterial genus Moorea and the co-evolution of its enzyme cassettes that create metabolic diversity. Progress in the development of heterologous expression systems for cyanobacterial gene clusters along with chemoenzymatic synthesis makes it possible to create new analogs. Additionally, phylum-wide genome sequencing projects have enhanced the discovery rate of new natural products and their distinctive enzymatic reactions. Summarizing, cyanobacterial biosynthetic gene clusters encode for a large toolbox of novel enzymes that catalyze unique chemical reactions, some of which may be useful in synthetic biology.

  13. New serine-derived gemini surfactants as gene delivery systems.

    PubMed

    Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; de Lima, Maria C Pedroso; Jurado, Amália S

    2015-01-01

    Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini surfactants combine the special aggregation properties characteristic of the gemini surfactants with high biocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differing in alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amide and ester), were evaluated for their ability to mediate gene delivery either per se or in combination with helper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic diameter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, able to transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA charge ratios (1/1-2/1). The most efficient complexes presented sizes suitable for intravenous administration and negative surface charge, a feature known to preclude potentially adverse interactions with serum components. This work brings forward a new family of gemini surfactants with great potential as gene delivery systems.

  14. Cleavage of Eukaryotic Translation Initiation Factor 4G by Exogenously Added Hybrid Proteins Containing Poliovirus 2Apro in HeLa Cells: Effects on Gene Expression

    PubMed Central

    Novoa, Isabel; Carrasco, Luis

    1999-01-01

    Efficient cleavage of both forms of eukaryotic initiation factor 4G (eIF4G-1 and eIF4G-2) has been achieved in HeLa cells by incubation with hybrid proteins containing poliovirus 2Apro. Entry of these proteins into cells is promoted by adenovirus particles. Substantial levels of ongoing translation on preexisting cellular mRNAs still continue for several hours after eIF4G degradation. Treatment of control HeLa cells with hypertonic medium causes an inhibition of translation that is reversed upon restoration of cells to normal medium. Protein synthesis is not restored in cells lacking intact eIF4G after hypertonic treatment. Notably, induction of synthesis of heat shock proteins still occurs in cells pretreated with poliovirus 2Apro, suggesting that transcription and translation of these mRNAs takes place even in the presence of cleaved eIF4G. Finally, the synthesis of luciferase was examined in a HeLa cell line bearing the luciferase gene under control of a tetracycline-regulated promoter. Transcription of the luciferase gene and transport of the mRNA to the cytoplasm occurs at control levels in eIF4G-deficient cells. However, luciferase synthesis is strongly inhibited in these cells. These findings indicate that intact eIF4G is necessary for the translation of mRNAs not engaged in translation with the exception of heat shock mRNAs but is not necessary for the translation of mRNAs that are being translated. PMID:10082510

  15. Changing ideas about eukaryotic origins.

    PubMed

    Williams, Tom A; Embley, T Martin

    2015-09-26

    The origin of eukaryotic cells is one of the most fascinating challenges in biology, and has inspired decades of controversy and debate. Recent work has led to major upheavals in our understanding of eukaryotic origins and has catalysed new debates about the roles of endosymbiosis and gene flow across the tree of life. Improved methods of phylogenetic analysis support scenarios in which the host cell for the mitochondrial endosymbiont was a member of the Archaea, and new technologies for sampling the genomes of environmental prokaryotes have allowed investigators to home in on closer relatives of founding symbiotic partners. The inference and interpretation of phylogenetic trees from genomic data remains at the centre of many of these debates, and there is increasing recognition that trees built using inadequate methods can prove misleading, whether describing the relationship of eukaryotes to other cells or the root of the universal tree. New statistical approaches show promise for addressing these questions but they come with their own computational challenges. The papers in this theme issue discuss recent progress on the origin of eukaryotic cells and genomes, highlight some of the ongoing debates, and suggest possible routes to future progress.

  16. Changing ideas about eukaryotic origins

    PubMed Central

    Williams, Tom A.; Embley, T. Martin

    2015-01-01

    The origin of eukaryotic cells is one of the most fascinating challenges in biology, and has inspired decades of controversy and debate. Recent work has led to major upheavals in our understanding of eukaryotic origins and has catalysed new debates about the roles of endosymbiosis and gene flow across the tree of life. Improved methods of phylogenetic analysis support scenarios in which the host cell for the mitochondrial endosymbiont was a member of the Archaea, and new technologies for sampling the genomes of environmental prokaryotes have allowed investigators to home in on closer relatives of founding symbiotic partners. The inference and interpretation of phylogenetic trees from genomic data remains at the centre of many of these debates, and there is increasing recognition that trees built using inadequate methods can prove misleading, whether describing the relationship of eukaryotes to other cells or the root of the universal tree. New statistical approaches show promise for addressing these questions but they come with their own computational challenges. The papers in this theme issue discuss recent progress on the origin of eukaryotic cells and genomes, highlight some of the ongoing debates, and suggest possible routes to future progress. PMID:26323752

  17. Harbinger transposons and an ancient HARBI1 gene derived from a transposase.

    PubMed

    Kapitonov, Vladimir V; Jurka, Jerzy

    2004-05-01

    In this study we report main properties of Harbinger DNA transposons identified in protists, plants, insects, worms, and vertebrates. This is the first superfamily of eukaryotic DNA transposons where all autonomous transposons, even those that are hosted by species from different kingdoms, encode two proteins: a superfamily-specific transposase and a DNA-binding protein characterized by the presence of the conserved SANT/myb/trihelix motif. The last motif is known to be important for the DNA binding by different transcription regulators. Therefore, we suggest that this protein is necessary for coordinated expression of the Harbinger transposase. Although mammalian genomes are free of recognizable remnants of Harbingers, we identified a widely expressed HARBI1 gene encoding a 350-aa protein entirely derived from a Harbinger transposase some 450-500 million years ago. The HARBI1 proteins are conserved in humans, rats, mice, cows, pigs, chickens, frogs, and various bony fish, as well as other extremely important proteins, including RAG1 and RAG2. Conserved motifs detected in the Harbinger transposases are also well preserved in the HARBI1 proteins. Therefore, the HARBI1 proteins are expected to be nucleases important for functioning of bony vertebrates. We also found that the protein most similar to HARBI1 is encoded by an autonomous Harbinger 3_DR transposon that was transpositionally active in the zebrafish genome a few million years ago. Nonautonomous transposons derived from Harbinger3_DR are characterized by a striking preference for a 17-bp target site never seen previously in any other DNA transposon. Based on this observation, we suggest that the hypothetical HARBI1 nucleases are also characterized by a strong DNA-target specificity.

  18. Endogenous Synthesis of Coenzyme Q in Eukaryotes

    PubMed Central

    Tran, UyenPhuong C.; Clarke, Catherine F.

    2007-01-01

    Coenzyme Q (Q) functions in the mitochondrial respiratory chain and serves as a lipophilic antioxidant. There is increasing interest in the use of Q as a nutritional supplement. Although the physiological significance of Q is extensively investigated in eukaryotes, ranging from yeast to human, the eukaryotic Q biosynthesis pathway is best characterized in the budding yeast Saccharomyces cerevisiae. At least ten genes (COQ1-COQ10) have been shown to be required for Q biosynthesis and function in respiration. This review highlights recent knowledge about the endogenous synthesis of Q in eukaryotes, with emphasis on S. cerevisiae as a model system. PMID:17482885

  19. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    NASA Astrophysics Data System (ADS)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  20. Comparative study of the validity of three regions of the 18S-rRNA gene for massively parallel sequencing-based monitoring of the planktonic eukaryote community.

    PubMed

    Tanabe, Akifumi S; Nagai, Satoshi; Hida, Kohsuke; Yasuike, Motoshige; Fujiwara, Atushi; Nakamura, Yoji; Takano, Yoshihito; Katakura, Seiji

    2016-03-01

    The nuclear 18S-rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)-based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S-rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1-3, V4-5 and V7-9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS-based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS-based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4-5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1-3 region, followed by the V7-9 region, and the lowest variability in the V4-5 region. The results of the MPS-based environmental surveys showed significantly higher identification power in the V1-3 and V7-9 regions than in the V4-5 region, but no significant difference was detected between the V1-3 and V7-9 regions. We therefore conclude that the V1-3 region will be the most suitable for future MPS-based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.

  1. Structure and function of eukaryotic chromosomes

    SciTech Connect

    Hennig, W.

    1987-01-01

    Contents: Introduction; Polytene Chromosomel Giant Chromosomes in Ciliates; The sp-I Genes in the Balbiani Rings of Chironomus Salivary Glands; The White Locus of Drosophila Melanogaster; The Genetic and Molecular Organization of the Dense Cluster of Functionally Related Vital Genes in the DOPA Decarboxylase Region of the Drosophila melanogaster Genome; Heat Shock Puffs and Response to Environmental Stress; The Y Chromosomal Lampbrush Loops of Drosophila; Contributions of Electron Microscopic Spreading Preparations (''Miller Spreads'') to the Analysis of Chromosome Structure; Replication of DNA in Eukaryotic Chromosomes; Gene Amplification in Dipteran Chromosomes; The Significance of Plant Transposable Elements in Biologically Relevant Processes; Arrangement of Chromosomes in Interphase Cell Nuclei; Heterochromatin and the Phenomenon of Chromosome Banding; Multiple Nonhistone Protein-DNA Complexes in Chromatin Regulate the Cell- and Stage-Specific Activity of an Eukaryotic Gene; Genetics of Sex Determination in Eukaryotes; Application of Basic Chromosome Research in Biotechnology and Medicine. This book presents an overview of various aspects of chromosome research.

  2. A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote, Trichomonas vaginalis.

    PubMed

    Markos, A; Miretsky, A; Müller, M

    1993-12-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), localized in the cytosol of Trichomonas vaginalis, was partially purified. The enzyme is specific for NAD+ and is similar in most of its catalytic properties to glycolytic GAPDHs from other organisms. Its sensitivity to koningic acid is similar to levels observed in GAPDHs from eubacteria and two orders of magnitude lower than those observed for eukaryotic GAPDHs. The complete amino acid sequence of T. vaginalis GAPDH was derived from the N-terminal sequence of the purified protein and the deduced sequence of a cDNA clone. It showed great similarity to other eubacterial and eukaryotic GAPDH sequences. The sequence of the S-loop displayed a eubacterial signature. The overall sequence was more similar to eubacterial sequences than to cytosolic and glycosomal eukaryotic sequences. In phylogenetic trees obtained with distance matrix and parsimony methods T. vaginalis GAPDH clustered with its eubacterial homologs. GAPDHs of other amitochondriate protists, belonging to early branches of the eukaryotic lineage (Giardia lamblia and Entamoeba histolytica--Smith M.W. and Doolittle R.F., unpublished data in GenBank), showed typical eukaryotic signatures and clustered with other eukaryotic sequences, indicating that T. vaginalis GAPDH occupies an anomalous position, possibly due to horizontal gene transfer from a eubacterium.

  3. Exaptive origins of regulated mRNA decay in eukaryotes

    PubMed Central

    Hamid, Fursham M.

    2016-01-01

    Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

  4. tRNA-Derived Fragments (tRFs): Emerging New Roles for an Ancient RNA in the Regulation of Gene Expression

    PubMed Central

    Keam, Simon P.; Hutvagner, Gyorgy

    2015-01-01

    This review will summarise the recent discoveries and current state of research on short noncoding RNAs derived from tRNAs—known as tRNA-derived fragments (tRFs). It will describe the features of the known subtypes of these RNAs; including sequence characteristics, protein interactors, expression characteristics, biogenesis, and similarity to canonical miRNA pathways. Also their role in regulating gene expression; including mediating translational suppression, will be discussed. We also highlight their potential use as biomarkers, functions in gene regulation and links to disease. Finally, this review will speculate as to the origin and rationale for the conservation of this novel class of noncoding RNAs amongst both prokaryotes and eukaryotes. PMID:26703738

  5. Circular code motifs in genomes of eukaryotes.

    PubMed

    El Soufi, Karim; Michel, Christian J

    2016-11-01

    A set X of 20 trinucleotides was identified in genes of bacteria, eukaryotes, plasmids and viruses, which has in average the highest occurrence in reading frame compared to its two shifted frames (Michel, 2015; Arquès and Michel, 1996). This set X has an interesting mathematical property as X is a circular code (Arquès and Michel, 1996). Thus, the motifs from this circular code X, called X motifs, have the property to always retrieve, synchronize and maintain the reading frame in genes. In this paper, we develop several statistical analyzes of X motifs in 138 available complete genomes of eukaryotes in which genes as well as non-gene regions are examined. Large X motifs (with lengths of at least 15 consecutive trinucleotides of X and compositions of at least 10 different trinucleotides of X among 20) have the highest occurrence in genomes of eukaryotes compared to its 23 large bijective motifs, its two large permuted motifs and large random motifs. The largest X motifs identified in eukaryotic genomes are presented, e.g. an X motif in a non-gene region of the genome Solanum pennellii with a length of 155 trinucleotides (465 nucleotides) and an expectation E=10(-71). In the human genome, the largest X motif occurs in a non-gene region of the chromosome 13 with a length of 36 trinucleotides and an expectation E=10(-11). X motifs in non-gene regions of genomes could be evolutionary relics of primitive genes using the circular code for translation. However, the proportion of X motifs (with lengths of at least 10 consecutive trinucleotides of X and compositions of at least 5 different trinucleotides of X among 20) in genes/non-genes of the 138 complete eukaryotic genomes is about 8. Thus, the X motifs occur preferentially in genes, as expected from the previous works of 20 years.

  6. The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

    SciTech Connect

    Fritz-Laylin, Lillian K.; Prochnik, Simon E.; Ginger, Michael L.; Dacks, Joel; Carpenter, Meredith L.; Field, Mark C.; Kuo, Alan; Paredez, Alex; Chapman, Jarrod; Pham, Jonathan; Shu, Shengqiang; Neupane, Rochak; Cipriano, Michael; Mancuso, Joel; Tu, Hank; Salamov, Asaf; Lindquist, Erika; Shapiro, Harris; Lucas, Susan; Grigoriev, Igor V.; Cande, W. Zacheus; Fulton, Chandler; Rokhsar, Daniel S.; Dawson, Scott C.

    2010-03-01

    Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

  7. The genome of Naegleria gruberi illuminates early eukaryotic versatility.

    PubMed

    Fritz-Laylin, Lillian K; Prochnik, Simon E; Ginger, Michael L; Dacks, Joel B; Carpenter, Meredith L; Field, Mark C; Kuo, Alan; Paredez, Alex; Chapman, Jarrod; Pham, Jonathan; Shu, Shengqiang; Neupane, Rochak; Cipriano, Michael; Mancuso, Joel; Tu, Hank; Salamov, Asaf; Lindquist, Erika; Shapiro, Harris; Lucas, Susan; Grigoriev, Igor V; Cande, W Zacheus; Fulton, Chandler; Rokhsar, Daniel S; Dawson, Scott C

    2010-03-01

    Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

  8. Acidocalcisomes of eukaryotes.

    PubMed

    Docampo, Roberto; Huang, Guozhong

    2016-08-01

    Acidocalcisomes are organelles rich in polyphosphate and cations and acidified by proton pumps. Although they have also been described in prokaryotes they have been better characterized in unicellular and multicellular eukaryotes. Eukaryotic acidocalcisomes belong to the group of lysosome-related organelles. They have a variety of functions, from the storage of cations and phosphorus to calcium signaling, autophagy, osmoregulation, blood coagulation, and inflammation. Acidocalcisomes of several unicellular eukaryotes possess a variety of transporters, channels and pumps implying a large energetic requirement for their maintenance and suggesting other important functions waiting to be discovered. PMID:27125677

  9. A case study for effects of operational taxonomic units from intracellular endoparasites and ciliates on the eukaryotic phylogeny: phylogenetic position of the haptophyta in analyses of multiple slowly evolving genes.

    PubMed

    Nozaki, Hisayoshi; Yang, Yi; Maruyama, Shinichiro; Suzaki, Toshinobu

    2012-01-01

    Recent multigene phylogenetic analyses have contributed much to our understanding of eukaryotic phylogeny. However, the phylogenetic positions of various lineages within the eukaryotes have remained unresolved or in conflict between different phylogenetic studies. These phylogenetic ambiguities might have resulted from mixtures or integration from various factors including limited taxon sampling, missing data in the alignment, saturations of rapidly evolving genes, mixed analyses of short- and long-branched operational taxonomic units (OTUs), intracellular endoparasite and ciliate OTUs with unusual substitution etc. In order to evaluate the effects from intracellular endoparasite and ciliate OTUs co-analyzed on the eukaryotic phylogeny and simplify the results, we here used two different sets of data matrices of multiple slowly evolving genes with small amounts of missing data and examined the phylogenetic position of the secondary photosynthetic chromalveolates Haptophyta, one of the most abundant groups of oceanic phytoplankton and significant primary producers. In both sets, a robust sister relationship between Haptophyta and SAR (stramenopiles, alveolates, rhizarians, or SA [stramenopiles and alveolates]) was resolved when intracellular endoparasite/ciliate OTUs were excluded, but not in their presence. Based on comparisons of character optimizations on a fixed tree (with a clade composed of haptophytes and SAR or SA), disruption of the monophyly between haptophytes and SAR (or SA) in the presence of intracellular endoparasite/ciliate OTUs can be considered to be a result of multiple evolutionary reversals of character positions that supported the synapomorphy of the haptophyte and SAR (or SA) clade in the absence of intracellular endoparasite/ciliate OTUs.

  10. The Eukaryotic Replisome Goes Under the Microscope.

    PubMed

    O'Donnell, Mike; Li, Huilin

    2016-03-21

    The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic replisome components include the Mcm2-7 complex, the CMG helicase, DNA polymerases, a Ctf4 trimer hub and the first look at a core replisome of 20 different proteins containing the helicase, primase, leading polymerase and a lagging strand polymerase. The eukaryotic core replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below. Additionally, structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the replisome in handling nucleosomes, which are important to DNA organization and gene regulation. This review provides a summary of some of the many recent advances in the structure of the eukaryotic replisome.

  11. Eukaryotic diversity in historical soil samples.

    PubMed

    Moon-van der Staay, Seung Yeo; Tzeneva, Vesela A; van der Staay, Georg W M; de Vos, Willem M; Smidt, Hauke; Hackstein, Johannes H P

    2006-09-01

    The eukaryotic biodiversity in historical air-dried samples of Dutch agricultural soil has been assessed by random sequencing of an 18S rRNA gene library and by denaturing gradient gel electrophoresis. Representatives of nearly all taxa of eukaryotic soil microbes could be identified, demonstrating that it is possible to study eukaryotic microbiota in samples from soil archives that have been stored for more than 30 years at room temperature. In a pilot study, 41 sequences were retrieved that could be assigned to fungi and a variety of aerobic and anaerobic protists such as cercozoans, ciliates, xanthophytes (stramenopiles), heteroloboseans, and amoebozoans. A PCR-denaturing gradient gel electrophoresis analysis of samples collected between 1950 and 1975 revealed significant changes in the composition of the eukaryotic microbiota.

  12. The Eukaryotic Replisome Goes Under the Microscope

    PubMed Central

    O’Donnell, Mike; Li, Huilin

    2016-01-01

    The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic replisome components include the Mcm2-7 complex, the CMG helicase, DNA polymerases, a Ctf4 trimer hub and the first look at a core replisome of 20 different proteins containing the helicase, primase, leading polymerase and a lagging strand polymerase. The eukaryotic core replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below. Additionally, structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the replisome in handling nucleosomes, which are important to DNA organization and gene regulation. This review provides a summary of some of the many recent advances in the structure of the eukaryotic replisome. PMID:27003891

  13. Eukaryotic tRNA paradox.

    PubMed

    Mitra, Sanga; Samadder, Arpa; Das, Pijush; Das, Smarajit; Chakrabarti, Jayprokas

    2015-01-01

    tRNAs are widely believed to segregate into two classes, I and II. Computational analysis of eukaryotic tRNA entries in Genomic tRNA Database, however, leads to new, albeit paradoxical, presence of more than a thousand class-I tRNAs with uncharacteristic long variable arms (V-arms), like in class-II. Out of 62,202 tRNAs from 69 eukaryotes, as many as 1431 class-I tRNAs have these novel extended V-arms, and we refer to them as paradoxical tRNAs (pxtRNAs). A great majority of these 1431 pxtRNA genes are located in intergenic regions, about 18% embedded in introns of genes or ESTs, and just one in 3'UTR. A check on the conservations of 2D and 3D base pairs for each position of these pxtRNAs reveals a few variations, but they seem to have almost all the known features (already known identity and conserved elements of tRNA). Analyses of the A-Box and B-Box of these pxtRNA genes in eukaryotes display salient deviations from the previously annotated conserved features of the standard promoters, whereas the transcription termination signals are just canonical and non-canonical runs of thymidine, similar to the ones in standard tRNA genes. There is just one such pxtRNA(ProAGG) gene in the entire human genome, and the availability of data allows epigenetic analysis of this human pxtRNA(ProAGG) in three different cell lines, H1 hESC, K562, and NHEK, to assess the level of its expression. Histone acetylation and methylation of this lone pxtRNA(ProAGG) gene in human differ from that of the nine standard human tRNA(ProAGG) genes. The V-arm nucleotide sequences and their secondary structures in pxtRNA differ from that of class-II tRNA. Considering these differences, hypotheses of alternative splicing, non-canonical intron and gene transfer are examined to partially improve the Cove scores of these pxtRNAs and to critically question their antecedence and novelty. PMID:25692737

  14. Structural disorder in eukaryotes.

    PubMed

    Pancsa, Rita; Tompa, Peter

    2012-01-01

    Based on early bioinformatic studies on a handful of species, the frequency of structural disorder of proteins is generally thought to be much higher in eukaryotes than in prokaryotes. To refine this view, we present here a comparative prediction study and analysis of 194 fully described eukaryotic proteomes and 87 reference prokaryotes for structural disorder. We found that structural disorder does distinguish eukaryotes from prokaryotes, but its frequency spans a very wide range in the two superkingdoms that largely overlap. The number of disordered binding regions and different Pfam domain types also contribute to distinguish eukaryotes from prokaryotes. Unexpectedly, the highest levels--and highest variability--of predicted disorder is found in protists, i.e. single-celled eukaryotes, often surpassing more complex eukaryote organisms, plants and animals. This trend contrasts with that of the number of domain types, which increases rather monotonously toward more complex organisms. The level of structural disorder appears to be strongly correlated with lifestyle, because some obligate intracellular parasites and endosymbionts have the lowest levels, whereas host-changing parasites have the highest level of predicted disorder. We conclude that protists have been the evolutionary hot-bed of experimentation with structural disorder, in a period when structural disorder was actively invented and the major functional classes of disordered proteins established.

  15. Evidence for lateral gene transfer (LGT) in the evolution of eubacteria-derived small GTPases in plant organelles

    PubMed Central

    Suwastika, I. Nengah; Denawa, Masatsugu; Yomogihara, Saki; Im, Chak Han; Bang, Woo Young; Ohniwa, Ryosuke L.; Bahk, Jeong Dong; Takeyasu, Kunio; Shiina, Takashi

    2014-01-01

    The genomes of free-living bacteria frequently exchange genes via lateral gene transfer (LGT), which has played a major role in bacterial evolution. LGT also played a significant role in the acquisition of genes from non-cyanobacterial bacteria to the lineage of “primary” algae and land plants. Small GTPases are widely distributed among prokaryotes and eukaryotes. In this study, we inferred the evolutionary history of organelle-targeted small GTPases in plants. Arabidopsis thaliana contains at least one ortholog in seven subfamilies of OBG-HflX-like and TrmE-Era-EngA-YihA-Septin-like GTPase superfamilies (together referred to as Era-like GTPases). Subcellular localization analysis of all Era-like GTPases in Arabidopsis revealed that all 30 eubacteria-related GTPases are localized to chloroplasts and/or mitochondria, whereas archaea-related DRG and NOG1 are localized to the cytoplasm and nucleus, respectively, suggesting that chloroplast- and mitochondrion-localized GTPases are derived from the ancestral cyanobacterium and α-proteobacterium, respectively, through endosymbiotic gene transfer (EGT). However, phylogenetic analyses revealed that plant organelle GTPase evolution is rather complex. Among the eubacterium-related GTPases, only four localized to chloroplasts (including one dual targeting GTPase) and two localized to mitochondria were derived from cyanobacteria and α-proteobacteria, respectively. Three other chloroplast-targeted GTPases were related to α-proteobacterial proteins, rather than to cyanobacterial GTPases. Furthermore, we found that four other GTPases showed neither cyanobacterial nor α-proteobacterial affiliation. Instead, these GTPases were closely related to clades from other eubacteria, such as Bacteroides (Era1, EngB-1, and EngB-2) and green non-sulfur bacteria (HflX). This study thus provides novel evidence that LGT significantly contributed to the evolution of organelle-targeted Era-like GTPases in plants. PMID:25566271

  16. Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal).

    PubMed

    Teppa, Roxana E; Petit, Daniel; Plechakova, Olga; Cogez, Virginie; Harduin-Lepers, Anne

    2016-01-01

    Cell surface of eukaryotic cells is covered with a wide variety of sialylated molecules involved in diverse biological processes and taking part in cell-cell interactions. Although the physiological relevance of these sialylated glycoconjugates in vertebrates begins to be deciphered, the origin and evolution of the genetic machinery implicated in their biosynthetic pathway are poorly understood. Among the variety of actors involved in the sialylation machinery, sialyltransferases are key enzymes for the biosynthesis of sialylated molecules. This review focus on β-galactoside α2,3/6-sialyltransferases belonging to the ST3Gal and ST6Gal families. We propose here an outline of the evolutionary history of these two major ST families. Comparative genomics, molecular phylogeny and structural bioinformatics provided insights into the functional innovations in sialic acid metabolism and enabled to explore how ST-gene function evolved in vertebrates. PMID:27517905

  17. Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal)

    PubMed Central

    Teppa, Roxana E.; Petit, Daniel; Plechakova, Olga; Cogez, Virginie; Harduin-Lepers, Anne

    2016-01-01

    Cell surface of eukaryotic cells is covered with a wide variety of sialylated molecules involved in diverse biological processes and taking part in cell–cell interactions. Although the physiological relevance of these sialylated glycoconjugates in vertebrates begins to be deciphered, the origin and evolution of the genetic machinery implicated in their biosynthetic pathway are poorly understood. Among the variety of actors involved in the sialylation machinery, sialyltransferases are key enzymes for the biosynthesis of sialylated molecules. This review focus on β-galactoside α2,3/6-sialyltransferases belonging to the ST3Gal and ST6Gal families. We propose here an outline of the evolutionary history of these two major ST families. Comparative genomics, molecular phylogeny and structural bioinformatics provided insights into the functional innovations in sialic acid metabolism and enabled to explore how ST-gene function evolved in vertebrates. PMID:27517905

  18. Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

    SciTech Connect

    Lykidis, Athanasios

    2006-12-01

    Phospholipid biosynthetic enzymes produce diverse molecular structures and are often present in multiple forms encoded by different genes. This work utilizes comparative genomics and phylogenetics for exploring the distribution, structure and evolution of phospholipid biosynthetic genes and pathways in 26 eukaryotic genomes. Although the basic structure of the pathways was formed early in eukaryotic evolution, the emerging picture indicates that individual enzyme families followed unique evolutionary courses. For example, choline and ethanolamine kinases and cytidylyltransferases emerged in ancestral eukaryotes, whereas, multiple forms of the corresponding phosphatidyltransferases evolved mainly in a lineage specific manner. Furthermore, several unicellular eukaryotes maintain bacterial-type enzymes and reactions for the synthesis of phosphatidylglycerol and cardiolipin. Also, base-exchange phosphatidylserine synthases are widespread and ancestral enzymes. The multiplicity of phospholipid biosynthetic enzymes has been largely generated by gene expansion in a lineage specific manner. Thus, these observations suggest that phospholipid biosynthesis has been an actively evolving system. Finally, comparative genomic analysis indicates the existence of novel phosphatidyltransferases and provides a candidate for the uncharacterized eukaryotic phosphatidylglycerol phosphate phosphatase.

  19. Eukaryotic diversity in an anaerobic aquifer polluted with landfill leachate.

    PubMed

    Brad, Traian; Braster, Martin; van Breukelen, Boris M; van Straalen, Nico M; Röling, Wilfred F M

    2008-07-01

    Eukaryotes may influence pollutant degradation processes in groundwater ecosystems by activities such as predation on bacteria and recycling of nutrients. Culture-independent community profiling and phylogenetic analysis of 18S rRNA gene fragments, as well as culturing, were employed to obtain insight into the sediment-associated eukaryotic community composition in an anaerobic sandy aquifer polluted with landfill leachate (Banisveld, The Netherlands). The microeukaryotic community at a depth of 1 to 5 m below the surface along a transect downgradient (21 to 68 m) from the landfill and at a clean reference location was diverse. Fungal sequences dominated most clone libraries. The fungal diversity was high, and most sequences were sequences of yeasts of the Basidiomycota. Sequences of green algae (Chlorophyta) were detected in parts of the aquifer close (<30 m) to the landfill. The bacterium-predating nanoflagellate Heteromita globosa (Cercozoa) was retrieved in enrichments, and its sequences dominated the clone library derived from the polluted aquifer at a depth of 5 m at a location 21 m downgradient from the landfill. The number of culturable eukaryotes ranged from 10(2) to 10(3) cells/g sediment. Culture-independent quantification revealed slightly higher numbers. Groundwater mesofauna was not detected. We concluded that the food chain in this polluted aquifer is short and consists of prokaryotes and fungi as decomposers of organic matter and protists as primary consumers of the prokaryotes.

  20. Transfer of DNA from Bacteria to Eukaryotes.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2016-01-01

    Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen), Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium), or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs), the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer. PMID:27406565

  1. Transfer of DNA from Bacteria to Eukaryotes

    PubMed Central

    2016-01-01

    ABSTRACT Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen), Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium), or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs), the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer. PMID:27406565

  2. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  3. Extrachromosomal elements in lower eukaryotes:

    SciTech Connect

    Wickner, R.B.; Hinnebusch, A.; Lambowitz, A.M.; Gunsalus, I.C.; Hollaender, A.

    1986-01-01

    While most genes are chromosomal, the nonchromosomal genes have played a disproportionate role in molecular biology, in part because of their easy accessibility and in part because they represent the most mobile portion of a cell's genome. Fungi, yeasts, protozoa, slime molds, algae, and other single-celled nucleated species, have recently gained dramatic popularity with the development of transformation methods for Saccharomyces, Neurospora, Schizosaccharomyces, Dictyostelium, and others of this group. The realization that Saccharomyces has oncogenes, RNA tumor viruses, intervening sequences, and all the mitotic, mitochondrial, and other structures typical of so-called ''higher'' eukaryotic organisms has confirmed the use of such organisms as model systems. Their use in biotechnology also shows great promise. The study in lower eukaryotes of mitochondria and chloroplasts has yielded many insights into similar structures in higher organisms as well as many unexpected finds, such as mechanisms of intron excision and the biology of introns, RNA catalysis, variation of the genetic code, and mechanisms of protein import across membranes.

  4. Organelle fission in eukaryotes.

    PubMed

    Osteryoung, K W

    2001-12-01

    The cellular machineries that power chloroplast and mitochondrial division in eukaryotes carry out the topologically challenging job of constricting and severing these double-membraned organelles. Consistent with their endosymbiotic origins, mitochondria in protists and chloroplasts in photosynthetic eukaryotes have evolved organelle-targeted forms of FtsZ, the prokaryotic ancestor of tubulin, as key components of their fission complexes. In fungi, animals and plants, mitochondria no longer utilize FtsZ for division, but several mitochondrial division proteins that localize to the outer membrane and intermembrane space, including two related to the filament-forming dynamins, have been identified in yeast and animals. Although the reactions that mediate organelle division are not yet understood, recent progress in uncovering the constituents of the organelle division machineries promises rapid advancement in our understanding of the biochemical mechanisms underlying the distinct but related processes of chloroplast and mitochondrial division in eukaryotes.

  5. Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents.

    PubMed

    Low, Woon-Kai; Li, Jing; Zhu, Mingzhao; Kommaraju, Sai Shilpa; Shah-Mittal, Janki; Hull, Ken; Liu, Jun O; Romo, Daniel

    2014-01-01

    A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19-C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells. PMID:24359706

  6. Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents.

    PubMed

    Low, Woon-Kai; Li, Jing; Zhu, Mingzhao; Kommaraju, Sai Shilpa; Shah-Mittal, Janki; Hull, Ken; Liu, Jun O; Romo, Daniel

    2014-01-01

    A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19-C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.

  7. Evaluation of frozen tissue-derived prognostic gene expression signatures in FFPE colorectal cancer samples

    PubMed Central

    Zhu, Jing; Deane, Natasha G.; Lewis, Keeli B.; Padmanabhan, Chandrasekhar; Washington, M. Kay; Ciombor, Kristen K.; Timmers, Cynthia; Goldberg, Richard M.; Beauchamp, R. Daniel; Chen, Xi

    2016-01-01

    Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients. PMID:27623752

  8. Evaluation of frozen tissue-derived prognostic gene expression signatures in FFPE colorectal cancer samples.

    PubMed

    Zhu, Jing; Deane, Natasha G; Lewis, Keeli B; Padmanabhan, Chandrasekhar; Washington, M Kay; Ciombor, Kristen K; Timmers, Cynthia; Goldberg, Richard M; Beauchamp, R Daniel; Chen, Xi

    2016-01-01

    Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE samples. In this study, to evaluate the gene expression of frozen tissue-derived prognostic signatures in FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 FFPE CRC samples measured by both platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients. PMID:27623752

  9. Eukaryotic Cell Panorama

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2011-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. This report describes the scientific results that support an illustration of a eukaryotic cell, enlarged by one million times to show the distribution and arrangement of macromolecules. The panoramic cross section includes eight panels that extend…

  10. Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids.

    PubMed

    Shalchian-Tabrizi, Kamran; Skånseng, Marianne; Ronquist, Fredrik; Klaveness, Dag; Bachvaroff, Tsvetan R; Delwiche, Charles F; Botnen, Andreas; Tengs, Torstein; Jakobsen, Kjetill S

    2006-08-01

    Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the

  11. The voyage of the microbial eukaryote.

    PubMed

    Worden, Alexandra Z; Allen, Andrew E

    2010-10-01

    Although genome data from unicellular marine eukaryotes is sparse, sequences from several supergroups have initiated an era of genome-enabled research aimed at understanding gene function, evolution, and adaptation in non-traditional model protists. Trends in genomic content within and between different lineages are emerging, including phylogenetically anomalous patterns, sometimes resulting from horizontal gene transfer. Some such genes have nutrient uptake and metabolism roles suggesting that bacterial and eukaryotic microbes have similar cellular-mineral-environmental constraints. Many 'accessory genome' components are of unknown function, but low gene copy numbers combined with small genomes make protists ideal for systems biology. Cultured and uncultured protists are providing insights to ecology, ancestral features and the role of cooption in development of complex traits. Various protists harbor features important in sexuality and multicellularity once believed to have originated in metazoans or other multicellular taxa.

  12. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

  13. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.)

    PubMed Central

    Asha, Srinivasan; Soniya, Eppurath V.

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5′tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5′tRFs in the infected leaf and root. The abundance of 5′tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5′AlaCGC tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5′Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

  14. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    PubMed

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

  15. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    PubMed

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

  16. Evolutionary origins of metabolic compartmentalization in eukaryotes

    PubMed Central

    Martin, William

    2010-01-01

    Many genes in eukaryotes are acquisitions from the free-living antecedents of chloroplasts and mitochondria. But there is no evolutionary ‘homing device’ that automatically directs the protein product of a transferred gene back to the organelle of its provenance. Instead, the products of genes acquired from endosymbionts can explore all targeting possibilities within the cell. They often replace pre-existing host genes, or even whole pathways. But the transfer of an enzymatic pathway from one compartment to another poses severe problems: over evolutionary time, the enzymes of the pathway acquire their targeting signals for the new compartment individually, not in unison. Until the whole pathway is established in the new compartment, newly routed individual enzymes are useless, and their genes will be lost through mutation. Here it is suggested that pathways attain novel compartmentation variants via a ‘minor mistargeting’ mechanism. If protein targeting in eukaryotic cells possesses enough imperfection such that small amounts of entire pathways continuously enter novel compartments, selectable units of biochemical function would exist in new compartments, and the genes could become selected. Dual-targeting of proteins is indeed very common within eukaryotic cells, suggesting that targeting variation required for this minor mistargeting mechanism to operate exists in nature. PMID:20124349

  17. Reinitiation enhances reliable transcriptional responses in eukaryotes.

    PubMed

    Liu, Bo; Yuan, Zhanjiang; Aihara, Kazuyuki; Chen, Luonan

    2014-08-01

    Gene transcription is a noisy process carried out by the transcription machinery recruited to the promoter. Noise reduction is a fundamental requirement for reliable transcriptional responses which in turn are crucial for signal transduction. Compared with the relatively simple transcription initiation in prokaryotes, eukaryotic transcription is more complex partially owing to its additional reinitiation mechanism. By theoretical analysis, we showed that reinitiation reduces noise in eukaryotic transcription independent of the transcription level. Besides, a higher reinitiation rate enables a stable scaffold complex an advantage in noise reduction. Finally, we showed that the coupling between scaffold formation and transcription can further reduce transcription noise independent of the transcription level. Furthermore, compared with the reinitiation mechanism, the noise reduction effect of the coupling can be of more significance in the case that the transcription level is low and the intrinsic noise dominates. Our results uncover a mechanistic route which eukaryotes may use to facilitate a more reliable response in the noisy transcription process. PMID:24850905

  18. Mitochondrion-related organelles in eukaryotic protists.

    PubMed

    Shiflett, April M; Johnson, Patricia J

    2010-01-01

    The discovery of mitochondrion-type genes in organisms thought to lack mitochondria led to the demonstration that hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as hydrogenosomes, mitosomes, or mitochondrion-like organelles, are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite their diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. This review examines the metabolism and biogenesis of these organelles in divergent unicellular microbes, with a focus on parasitic protists.

  19. Eukaryotic association module in phage WO genomes from Wolbachia

    PubMed Central

    Bordenstein, Sarah R.; Bordenstein, Seth R.

    2016-01-01

    Viruses are trifurcated into eukaryotic, archaeal and bacterial categories. This domain-specific ecology underscores why eukaryotic viruses typically co-opt eukaryotic genes and bacteriophages commonly harbour bacterial genes. However, the presence of bacteriophages in obligate intracellular bacteria of eukaryotes may promote DNA transfers between eukaryotes and bacteriophages. Here we report a metagenomic analysis of purified bacteriophage WO particles of Wolbachia and uncover a eukaryotic association module in the complete WO genome. It harbours predicted domains, such as the black widow latrotoxin C-terminal domain, that are uninterrupted in bacteriophage genomes, enriched with eukaryotic protease cleavage sites and combined with additional domains to forge one of the largest bacteriophage genes to date (14,256 bp). To the best of our knowledge, these eukaryotic-like domains have never before been reported in packaged bacteriophages and their phylogeny, distribution and sequence diversity imply lateral transfers between bacteriophage/prophage and animal genomes. Finally, the WO genome sequences and identification of attachment sites will potentially advance genetic manipulation of Wolbachia. PMID:27727237

  20. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa.

    PubMed

    Cavalier-Smith, T

    2002-03-01

    Eukaryotes and archaebacteria form the clade neomura and are sisters, as shown decisively by genes fragmented only in archaebacteria and by many sequence trees. This sisterhood refutes all theories that eukaryotes originated by merging an archaebacterium and an alpha-proteobacterium, which also fail to account for numerous features shared specifically by eukaryotes and actinobacteria. I revise the phagotrophy theory of eukaryote origins by arguing that the essentially autogenous origins of most eukaryotic cell properties (phagotrophy, endomembrane system including peroxisomes, cytoskeleton, nucleus, mitosis and sex) partially overlapped and were synergistic with the symbiogenetic origin of mitochondria from an alpha-proteobacterium. These radical innovations occurred in a derivative of the neomuran common ancestor, which itself had evolved immediately prior to the divergence of eukaryotes and archaebacteria by drastic alterations to its eubacterial ancestor, an actinobacterial posibacterium able to make sterols, by replacing murein peptidoglycan by N-linked glycoproteins and a multitude of other shared neomuran novelties. The conversion of the rigid neomuran wall into a flexible surface coat and the associated origin of phagotrophy were instrumental in the evolution of the endomembrane system, cytoskeleton, nuclear organization and division and sexual life-cycles. Cilia evolved not by symbiogenesis but by autogenous specialization of the cytoskeleton. I argue that the ancestral eukaryote was uniciliate with a single centriole (unikont) and a simple centrosomal cone of microtubules, as in the aerobic amoebozoan zooflagellate Phalansterium. I infer the root of the eukaryote tree at the divergence between opisthokonts (animals, Choanozoa, fungi) with a single posterior cilium and all other eukaryotes, designated 'anterokonts' because of the ancestral presence of an anterior cilium. Anterokonts comprise the Amoebozoa, which may be ancestrally unikont, and a vast

  1. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa.

    PubMed

    Cavalier-Smith, T

    2002-03-01

    Eukaryotes and archaebacteria form the clade neomura and are sisters, as shown decisively by genes fragmented only in archaebacteria and by many sequence trees. This sisterhood refutes all theories that eukaryotes originated by merging an archaebacterium and an alpha-proteobacterium, which also fail to account for numerous features shared specifically by eukaryotes and actinobacteria. I revise the phagotrophy theory of eukaryote origins by arguing that the essentially autogenous origins of most eukaryotic cell properties (phagotrophy, endomembrane system including peroxisomes, cytoskeleton, nucleus, mitosis and sex) partially overlapped and were synergistic with the symbiogenetic origin of mitochondria from an alpha-proteobacterium. These radical innovations occurred in a derivative of the neomuran common ancestor, which itself had evolved immediately prior to the divergence of eukaryotes and archaebacteria by drastic alterations to its eubacterial ancestor, an actinobacterial posibacterium able to make sterols, by replacing murein peptidoglycan by N-linked glycoproteins and a multitude of other shared neomuran novelties. The conversion of the rigid neomuran wall into a flexible surface coat and the associated origin of phagotrophy were instrumental in the evolution of the endomembrane system, cytoskeleton, nuclear organization and division and sexual life-cycles. Cilia evolved not by symbiogenesis but by autogenous specialization of the cytoskeleton. I argue that the ancestral eukaryote was uniciliate with a single centriole (unikont) and a simple centrosomal cone of microtubules, as in the aerobic amoebozoan zooflagellate Phalansterium. I infer the root of the eukaryote tree at the divergence between opisthokonts (animals, Choanozoa, fungi) with a single posterior cilium and all other eukaryotes, designated 'anterokonts' because of the ancestral presence of an anterior cilium. Anterokonts comprise the Amoebozoa, which may be ancestrally unikont, and a vast

  2. Atypical mitochondrial inheritance patterns in eukaryotes.

    PubMed

    Breton, Sophie; Stewart, Donald T

    2015-10-01

    Mitochondrial DNA (mtDNA) is predominantly maternally inherited in eukaryotes. Diverse molecular mechanisms underlying the phenomenon of strict maternal inheritance (SMI) of mtDNA have been described, but the evolutionary forces responsible for its predominance in eukaryotes remain to be elucidated. Exceptions to SMI have been reported in diverse eukaryotic taxa, leading to the prediction that several distinct molecular mechanisms controlling mtDNA transmission are present among the eukaryotes. We propose that these mechanisms will be better understood by studying the deviations from the predominating pattern of SMI. This minireview summarizes studies on eukaryote species with unusual or rare mitochondrial inheritance patterns, i.e., other than the predominant SMI pattern, such as maternal inheritance of stable heteroplasmy, paternal leakage of mtDNA, biparental and strictly paternal inheritance, and doubly uniparental inheritance of mtDNA. The potential genes and mechanisms involved in controlling mitochondrial inheritance in these organisms are discussed. The linkage between mitochondrial inheritance and sex determination is also discussed, given that the atypical systems of mtDNA inheritance examined in this minireview are frequently found in organisms with uncommon sexual systems such as gynodioecy, monoecy, or andromonoecy. The potential of deviations from SMI for facilitating a better understanding of a number of fundamental questions in biology, such as the evolution of mtDNA inheritance, the coevolution of nuclear and mitochondrial genomes, and, perhaps, the role of mitochondria in sex determination, is considerable.

  3. Sequencing our way towards understanding global eukaryotic biodiversity

    PubMed Central

    Bik, Holly M.; Porazinska, Dorota L.; Creer, Simon; Caporaso, J. Gregory; Knight, Rob; Thomas, W. Kelley

    2011-01-01

    Microscopic eukaryotes are abundant, diverse, and fill critical ecological roles across every ecosystem on earth, yet there is a well-recognized gap in our understanding of their global biodiversity. Fundamental advances in DNA sequencing and bioinformatics now allow accurate en masse biodiversity assessments of microscopic eukaryotes from environmental samples. Despite a promising outlook, the field of eukaryotic marker gene surveys faces significant challenges: how to generate data that is most useful to the community, especially in the face of evolving sequencing technology and bioinformatics pipelines, and how to incorporate an expanding number of target genes. PMID:22244672

  4. [The origin of the eukaryotic cell. III. Principles of the morphofunctional organization of the eukaryotic cell].

    PubMed

    Seravin, L N

    1986-08-01

    The eukaryotic plasmalemma, eukaryotic cytoplasm with its usual cytomembranes, and eukaryotic nucleus are obligatory components of the eukaryotic cell. All other structural elements (organelles) are only derivates of the aforesaid cell components and they may be absent sometimes. There are protozoans having simultaneously no flagelles, mitochondria and chloroplasts (all the representatives of phylum Microspora, amoeba Pelomyxa palustris, and others). The following five general principles play the main role in the morphofunctional organization of the cell. The principle of hierarchy of block organization of living systems. Complex morphofunctional blocks (organelles) specific for the eukaryotic cell are formed. The compartmentalization principle. The main cell organelles (nuclei, flagellae, mitochondria, chloroplasts, etc.) undergo a relative morphological isolation from each other and other cell organelles by means of the total or partial surrounding by membranes; this may ensure the originality of their evolution and function. The principle of poly- and oligomerization of morphofunctional blocks. It permits the cell to enlarge its sizes and to raise the level of integration. The principle of heterochrony, including three subprinciples: conservatism of useful signs; a strong acceleration of evolutionary development of the separate blocks; simplification of the structure, reduction or total disappearance of some blocks. It explains a preservation of prokaryotic signs in the eukaryotic cell or in its organelles. The principle of independent origin of similar morphofunctional blocks in the process of evolution of living systems. The parallelism of the signs in unrelated groups of cells (or protists) arises due to this principle.

  5. The small molecule '1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate' and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha.

    PubMed

    Hong, Mi-Na; Nam, Ky-Youb; Kim, Kyung Kon; Kim, So-Young; Kim, InKi

    2016-05-01

    By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

  6. Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

    PubMed

    Garg, Sriram G; Martin, William F

    2016-01-01

    Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically

  7. Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

    PubMed

    Garg, Sriram G; Martin, William F

    2016-07-02

    Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically

  8. Molecular machines encoded by bacterially-derived multi-domain gene fusions that potentially synthesize, N-methylate and transfer long chain polyamines in diatoms.

    PubMed

    Michael, Anthony J

    2011-09-01

    Silica glass formation in diatoms requires the biosynthesis of unusual, very long chain polyamines (LCPA) composed of iterated aminopropyl units. Diatoms processively synthesize LCPA, N-methylate the amine groups and transfer concatenated, N-dimethylated aminopropyl groups to silaffin proteins. Here I show that diatom genomes possess signal peptide-containing gene fusions of bacterially-derived polyamine biosynthetic enzymes S-adenosylmethionine decarboxylase (AdoMetDC) and an aminopropyltransferase, sometimes fused to a eukaryotic histone N-methyltransferase domain, that potentially synthesize and N-methylate LCPA. Fusions of similar, alternatively configured domains but with a catalytically dead AdoMetDC and in one case a Tudor domain, may N-dimethylate and transfer multiple aminopropyl unit polyamines onto silaffin proteins.

  9. Positive Regulation in a Eukaryote, a Study of the uaY Gene of ASPERGILLUS NIDULANS: I. Characterization of Alleles, Dominance and Complementation Studies, and a Fine Structure Map of the uaY -oxpA Cluster

    PubMed Central

    Scazzocchio, Claudio; Sdrin, Nicholas; Ong, Gloria

    1982-01-01

    In this paper we characterize genetically a positive eukaryotic regulatory gene: the uaY gene of the ascomycete Aspergillus nidulans. Several steps in the uptake and degradation of purines are under the control of the uaY gene (summarized in Scazzocchio and Gorton 1977). In the present paper 12 uaY- mutations are characterized with respect to their inducibility for adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase and by the absence of the uric acid-xanthine permease scored in vivo by resistance to 2-thiouric acid. While 10 mutations are uniformly unleaky, two others are almost wild type for the induction of urate oxidase. A fine structure map of the uaY gene shows that the two "leaky" mutations are not clustered. The fine structure mapping unambiguously positions six uaY alleles and provides preliminary but interesting trends regarding the pattern of gene conversion in the uaY gene. The enzyme levels in all uaY-/uaY+ heterozygous diploids are intermediate between the corresponding uaY-/uaY- and uaY+/uaY+ homozygous diploids, suggesting that one functional copy of the uaY gene is able to mediate the complete induction of only one set of structural genes. No complementation was found between any two uaY- alleles. This establishes that the mutations showing either of the phenotypes are alleles in the same gene; it fails to provide evidence for intracistronic complementation. A mutation, oxpA5, causes resistance to the xanthine analogue oxypurinol (4, 6-dihydroxypyrazolo-(3, 4-d)-pyrimidine) and partial constitutivity of adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase. The constitutive phenotype is suppressed by mutations blocking the synthesis of intracellular inducers. The mutation is recessive and complements fully with the 11 uaY- mutations tested. It maps to the left of all 12 uaY mutations to which it has been crossed. The data indicate that both the resistance and constitutivity arise from one

  10. Structural diversity of eukaryotic small subunit ribosomal RNAs. Evolutionary implications.

    PubMed

    Sogin, M L; Gunderson, J H

    1987-01-01

    The phylogenetic diversity of the eukaryotic kingdom was assessed by comparing the structural and evolutionary diversity of 18-20S ribosomal RNA genes. The coding regions for cytoplasmic small subunit ribosomal RNA genes vary in length from 1753 to 2305 nucleotides, and they appear to be evolutionary mosaics in which highly and partially conserved sequences are interspersed among regions that display very high rates of genetic drift. Structural similarities between these gene sequences were used to establish a phylogenetic framework for the eukaryotes. The extent of sequence variation within the eukaryotes exceeds that displayed within the eubacterial or archaebacterial lines of descent. The kinetoplastids and euglenoids represent the earliest branchings among the eukaryotes. These branchings preceded the divergence of lineages leading to the slime molds and apicomplexans and far antedate a radiative period that gave rise to the plants, animals, fungi, and other protists.

  11. Endosymbiotic theories for eukaryote origin.

    PubMed

    Martin, William F; Garg, Sriram; Zimorski, Verena

    2015-09-26

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe.

  12. Endosymbiotic theories for eukaryote origin

    PubMed Central

    Martin, William F.; Garg, Sriram; Zimorski, Verena

    2015-01-01

    For over 100 years, endosymbiotic theories have figured in thoughts about the differences between prokaryotic and eukaryotic cells. More than 20 different versions of endosymbiotic theory have been presented in the literature to explain the origin of eukaryotes and their mitochondria. Very few of those models account for eukaryotic anaerobes. The role of energy and the energetic constraints that prokaryotic cell organization placed on evolutionary innovation in cell history has recently come to bear on endosymbiotic theory. Only cells that possessed mitochondria had the bioenergetic means to attain eukaryotic cell complexity, which is why there are no true intermediates in the prokaryote-to-eukaryote transition. Current versions of endosymbiotic theory have it that the host was an archaeon (an archaebacterium), not a eukaryote. Hence the evolutionary history and biology of archaea increasingly comes to bear on eukaryotic origins, more than ever before. Here, we have compiled a survey of endosymbiotic theories for the origin of eukaryotes and mitochondria, and for the origin of the eukaryotic nucleus, summarizing the essentials of each and contrasting some of their predictions to the observations. A new aspect of endosymbiosis in eukaryote evolution comes into focus from these considerations: the host for the origin of plastids was a facultative anaerobe. PMID:26323761

  13. Deadenylation and its regulation in eukaryotic cells.

    PubMed

    Zhang, Xiaokan; Kleiman, Frida E; Devany, Emral

    2014-01-01

    Messenger RNA deadenylation is a process that allows rapid regulation of gene expression in response to different cellular conditions. The change of the mRNA poly(A) tail length by the activation of deadenylation might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. Activation of deadenylation processes are highly regulated and associated with different cellular conditions such as cancer, development, mRNA surveillance, DNA damage response, and cell differentiation. In the last few years, new technologies for studying deadenylation have been developed. Here we overview concepts related to deadenylation and its regulation in eukaryotic cells. We also describe some of the most commonly used protocols to study deadenylation in eukaryotic cells.

  14. Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

    PubMed

    Boussif, O; Delair, T; Brua, C; Veron, L; Pavirani, A; Kolbe, H V

    1999-01-01

    Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa. PMID:10502356

  15. The Diversity of Eukaryotes.

    PubMed

    Patterson

    1999-10-01

    The discipline of evolutionary protistology has emerged in the past 30 yr. There is as yet no agreed view of how protists are interrelated or how they should be classified. The foundations of a stable taxonomic superstructure for the protists and other eukaryotes lie in cataloging the diversity of the major monophyletic lineages of these organisms. The use of common patterns of cell organization (ultrastructural identity) seems to provide us with the most robust hypotheses of such lineages. These lineages are placed in 71 groups without identifiable sister taxa. These groups are here referred to as "major building blocks." For the first time, the compositions, ultrastructural identities, synapomorphies (where available), and subgroups of the major building blocks are summarized. More than 200 further lineages without clear identities are listed. This catalog includes all known major elements of the comprehensive evolutionary tree of protists and eukaryotes. Different approaches among protistologists to issues of nomenclature, ranking, and definitions of these groups are discussed, with particular reference to two groups-the stramenopiles and the Archezoa. The concept of "extended in-group" is introduced to refer to in-groups and the most proximate sister group and to assist in identifying the hierarchical location of taxa.

  16. [Computational approaches for identification and classification of transposable elements in eukaryotic genomes].

    PubMed

    Xu, Hong-En; Zhang, Hua-Hao; Han, Min-Jin; Shen, Yi-Hong; Huang, Xian-Zhi; Xiang, Zhong-Huai; Zhang, Ze

    2012-08-01

    Repetitive sequences (repeats) represent a significant fraction of the eukaryotic genomes and can be divided into tandem repeats, segmental duplications, and interspersed repeats on the basis of their sequence characteristics and how they are formed. Most interspersed repeats are derived from transposable elements (TEs). Eukaryotic TEs have been subdivided into two major classes according to the intermediate they use to move. The transposition and amplification of TEs have a great impact on the evolution of genes and the stability of genomes. However, identification and classification of TEs are complex and difficult due to the fact that their structure and classification are complex and diverse compared with those of other types of repeats. Here, we briefly introduced the function and classification of TEs, and summarized three different steps for identification, classification and annotation of TEs in eukaryotic genomes: (1) assembly of a repeat library, (2) repeat correction and classification, and (3) genome annotation. The existing computational approaches for each step were summarized and the advantages and disadvantages of the approaches were also highlighted in this review. To accurately identify, classify, and annotate the TEs in eukaryotic genomes requires combined methods. This review provides useful information for biologists who are not familiar with these approaches to find their way through the forest of programs.

  17. [The evolutionary fates of the flagellum from the unicellular ancestral eukaryote].

    PubMed

    Bornens, Michel

    2011-01-01

    The microtubule-based flagellum, or basal body/axoneme, is ancestral: it is a so-called "derived character" defining the extant eukaryote branch, present at the apparition of the early eukaryotic cell. It is a genuine cell compartment. Its structural conservation throughout evolution is remarkable, as well as the molecular conservation of the intraflagellar transport genes. From the start, it links cell sensation and cell locomotion, two properties which require integrated wiring at the cell level. The cell division machinery has been also tightly coupled with this organelle throughout evolution, possibly to ensure the continuity of this cellular polarized organization through division. The association of three basic cell modules, locomotion, sensation and division, all expressing a facet of cell polarity, within the same apparatus, could have given optimal survival value to primitive eukaryotic cells and could explain the evolutionary conservation and success of the basal body/axoneme. In vertebrates, primary cilia are critical sensory organelles, including during early mammalian development. Today the eukaryotic flagellum is not only an important issue for basic cell biology and developmental biology but also for human pathology. Many apparently unrelated severe developmental pathologies turned out to be all connected to the assembly or the function of the primary or beating cilia. The current efforts on human ciliopathies benefit directly from long lasting basic research on the flagellar assembly in experimental models like Chlamydomonas reinhardtii.

  18. Insights into the distribution and functions of the eukaryotic GPI-like anchored genes among Mycobacterium from a comparative genomic perspective.

    PubMed

    Deng, Wanyan; Zeng, Jie; Xiang, Xiaohong; Xie, Jianping

    2012-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins range from small peptides to larger antigens and fulfill a variety of cellular functions in eukaryotes. We speculated there should be such molecules in intracellular pathogens such as Mycobacterium due to their complex interplay with the host. However, no prior publications have touched this topic. To explore the existence and distribution of GPI-like molecules among Mycobacterium, we exhaustively analyzed all publicly available Mycobacterium genomes and found that the GPI-like signal sequences are prevalent among Mycobacterium, and a significant dichotomy between nonpathogenic Mycobacterium (exemplified by Mycobacterium smegmatis) and pathogenic Mycobacterium (exemplified by Mycobacterium tuberculosis), through genome-wide GPI-SOM analysis. Some well-documented anti-tuberculosis drug targets are predicted to have GPI-like anchored signals, such as KasA and atpE. Interestingly, Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins predicted to have GPI-anchoring sequence are unique to pathogenic Mycobacterium. These results can be further explored for better control measures against tuberculosis. PMID:23272800

  19. Energetics and genetics across the prokaryote-eukaryote divide

    PubMed Central

    2011-01-01

    Background All complex life on Earth is eukaryotic. All eukaryotic cells share a common ancestor that arose just once in four billion years of evolution. Prokaryotes show no tendency to evolve greater morphological complexity, despite their metabolic virtuosity. Here I argue that the eukaryotic cell originated in a unique prokaryotic endosymbiosis, a singular event that transformed the selection pressures acting on both host and endosymbiont. Results The reductive evolution and specialisation of endosymbionts to mitochondria resulted in an extreme genomic asymmetry, in which the residual mitochondrial genomes enabled the expansion of bioenergetic membranes over several orders of magnitude, overcoming the energetic constraints on prokaryotic genome size, and permitting the host cell genome to expand (in principle) over 200,000-fold. This energetic transformation was permissive, not prescriptive; I suggest that the actual increase in early eukaryotic genome size was driven by a heavy early bombardment of genes and introns from the endosymbiont to the host cell, producing a high mutation rate. Unlike prokaryotes, with lower mutation rates and heavy selection pressure to lose genes, early eukaryotes without genome-size limitations could mask mutations by cell fusion and genome duplication, as in allopolyploidy, giving rise to a proto-sexual cell cycle. The side effect was that a large number of shared eukaryotic basal traits accumulated in the same population, a sexual eukaryotic common ancestor, radically different to any known prokaryote. Conclusions The combination of massive bioenergetic expansion, release from genome-size constraints, and high mutation rate favoured a protosexual cell cycle and the accumulation of eukaryotic traits. These factors explain the unique origin of eukaryotes, the absence of true evolutionary intermediates, and the evolution of sex in eukaryotes but not prokaryotes. Reviewers This article was reviewed by: Eugene Koonin, William Martin

  20. Single-cell transcriptomics for microbial eukaryotes.

    PubMed

    Kolisko, Martin; Boscaro, Vittorio; Burki, Fabien; Lynn, Denis H; Keeling, Patrick J

    2014-11-17

    One of the greatest hindrances to a comprehensive understanding of microbial genomics, cell biology, ecology, and evolution is that most microbial life is not in culture. Solutions to this problem have mainly focused on whole-community surveys like metagenomics, but these analyses inevitably loose information and present particular challenges for eukaryotes, which are relatively rare and possess large, gene-sparse genomes. Single-cell analyses present an alternative solution that allows for specific species to be targeted, while retaining information on cellular identity, morphology, and partitioning of activities within microbial communities. Single-cell transcriptomics, pioneered in medical research, offers particular potential advantages for uncultivated eukaryotes, but the efficiency and biases have not been tested. Here we describe a simple and reproducible method for single-cell transcriptomics using manually isolated cells from five model ciliate species; we examine impacts of amplification bias and contamination, and compare the efficacy of gene discovery to traditional culture-based transcriptomics. Gene discovery using single-cell transcriptomes was found to be comparable to mass-culture methods, suggesting single-cell transcriptomics is an efficient entry point into genomic data from the vast majority of eukaryotic biodiversity.

  1. Functional Gene Networks: R/Bioc package to generate and analyse gene networks derived from functional enrichment and clustering

    PubMed Central

    Aibar, Sara; Fontanillo, Celia; Droste, Conrad; De Las Rivas, Javier

    2015-01-01

    Summary: Functional Gene Networks (FGNet) is an R/Bioconductor package that generates gene networks derived from the results of functional enrichment analysis (FEA) and annotation clustering. The sets of genes enriched with specific biological terms (obtained from a FEA platform) are transformed into a network by establishing links between genes based on common functional annotations and common clusters. The network provides a new view of FEA results revealing gene modules with similar functions and genes that are related to multiple functions. In addition to building the functional network, FGNet analyses the similarity between the groups of genes and provides a distance heatmap and a bipartite network of functionally overlapping genes. The application includes an interface to directly perform FEA queries using different external tools: DAVID, GeneTerm Linker, TopGO or GAGE; and a graphical interface to facilitate the use. Availability and implementation: FGNet is available in Bioconductor, including a tutorial. URL: http://bioconductor.org/packages/release/bioc/html/FGNet.html Contact: jrivas@usal.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25600944

  2. Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network.

    PubMed

    Qin, Tingting; Matmati, Nabil; Tsoi, Lam C; Mohanty, Bidyut K; Gao, Nan; Tang, Jijun; Lawson, Andrew B; Hannun, Yusuf A; Zheng, W Jim

    2014-10-01

    To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes' Ontology Fingerprints--a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms' corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general.

  3. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A {beta}-galactosidase reporter gene was inserted in place of the {beta}-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the {beta}-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal {beta}-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the {beta}-casein gene.

  4. Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins

    PubMed Central

    Koumandou, V Lila; Dacks, Joel B; Coulson, Richard MR; Field, Mark C

    2007-01-01

    Background In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery. Results We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common eukaryotic ancestor (LCEA

  5. MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

    DOE PAGESBeta

    Paugh, Steven W.; Coss, David R.; Bao, Ju; Laudermilk, Lucas T.; Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael Rex; et al

    2016-02-04

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10-16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

  6. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

    PubMed Central

    Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

    2016-01-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  7. [Codon optimization and eukaryotic expression analysis of the analgesic peptide gene BmK AngM1 from Buthus martensii Karsch].

    PubMed

    Yang, Jin-ling; Gao, Li-li; Zhu, Ping; Hou, Qi; Wang, Fen; Yu, Wen-bo; Nie, Tao

    2012-10-01

    Codon bias is an important factor which influences heterologous gene expression. Optimizing codon sequence could improve expression level of heterologous gene. In order to improve the expression level of BmK AngM1 gene encoding the analgesic peptide from Buthus martensii Karsch in Pichia pastoris, the codon-optimized BmK AngM1 gene according to its cDNA sequence and the preference codon usage of P. pastoris were cloned into expression vector pPIC9K and then transformed into P. pastoris. The expersion of recombinant BmK AngM1 (rBmK AngM1) was inducced by methanol in the medium, and the expression level of the optimized BmK AngM1 gene was 3.7 times of the native one. These results suggested that the expression of BmK AngM1 in P. pastoris could be successfully improved by codon optimization. PMID:23289154

  8. Secretive ciliates and putative asexuality in microbial eukaryotes.

    PubMed

    Dunthorn, Micah; Katz, Laura A

    2010-05-01

    Facultative sexuality is assumed to have occurred in the ancestor of all extant eukaryotes, but the distribution and maintenance of sex among microbial eukaryotes is still under debate. In this paper, we address the purported asexuality in colpodean ciliates as an exemplary lineage. Colpodeans are a primarily terrestrial clade thought to have arisen up to 900 MYA and contain one known derived sexual species. We conclude that the putative asexuality of this lineage is an observational artifact. We suggest that the same might hold for other microbial eukaryotes, and that many are secretively sexual as well. Theoretical work from the distantly related plants and animals suggests that both the evolutionary success of ancient asexuals and the reversal of the loss of sex are highly unlikely, further suggesting that colpodeans are secretively sexual. However, it remains to be seen to what extent sexual theories and predictions derived from macro-organismic lineages apply also to microbial eukaryotes.

  9. Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network

    PubMed Central

    Qin, Tingting; Matmati, Nabil; Tsoi, Lam C.; Mohanty, Bidyut K.; Gao, Nan; Tang, Jijun; Lawson, Andrew B.; Hannun, Yusuf A.; Zheng, W. Jim

    2014-01-01

    To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes’ Ontology Fingerprints—a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms’ corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities. We applied this method to the sphingolipid pathway in yeast and found that many top-ranked genes indeed displayed altered sphingolipid pathway functions, initially measured by their sensitivity to myriocin, an inhibitor of de novo sphingolipid biosynthesis. Further experiments confirmed the modulation of the sphingolipid pathway by one of these genes, PFA4, encoding a palmitoyl transferase. Comparative analysis showed that few of these novel genes could be discovered by other existing methods. Our novel gene network provides a unique and comprehensive resource to study pathway modulations and systems biology in general. PMID:25063300

  10. Eukaryotic diversity at pH extremes.

    PubMed

    Amaral-Zettler, Linda A

    2012-01-01

    Extremely acidic (pH < 3) and extremely alkaline (pH > 9) environments support a diversity of single-cell and to a lesser extent, multicellular eukaryotic life. This study compared alpha and beta diversity in eukaryotic communities from seven diverse aquatic environments with pH values ranging from 2 to 11 using massively-parallel pyrotag sequencing targeting the V9 hypervariable region of the 18S ribosomal RNA (rRNA) gene. A total of 946 operational taxonomic units (OTUs) were recovered at a 6% cut-off level (94% similarity) across the sampled environments. Hierarchical clustering of the samples segregated the communities into acidic and alkaline groups. Similarity percentage (SIMPER) analysis followed by indicator OTU analysis (IOA) and non-metric multidimensional scaling (NMDS) were used to determine which characteristic groups of eukaryotic taxa typify acidic or alkaline extremes and the extent to which pH explains eukaryotic community structure in these environments. Spain's Rio Tinto yielded the fewest observed OTUs while Nebraska Sandhills alkaline lakes yielded the most. Distinct OTUs, including metazoan OTUs, numerically dominated pH extreme sites. Indicator OTUs included the diatom Pinnularia and unidentified opisthokonts (Fungi and Filasterea) in the extremely acidic environments, and the ciliate Frontonia across the extremely alkaline sites. Inferred from NMDS, pH explained only a modest fraction of the variation across the datasets, indicating that other factors influence the underlying community structure in these environments. The findings from this study suggest that the ability for eukaryotes to adapt to pH extremes over a broad range of values may be rare, but further study of taxa that can broadly adapt across diverse acidic and alkaline environments, respectively present good models for understanding adaptation and should be targeted for future investigations.

  11. Eukaryotic diversity at pH extremes

    PubMed Central

    Amaral-Zettler, Linda A.

    2013-01-01

    Extremely acidic (pH < 3) and extremely alkaline (pH > 9) environments support a diversity of single-cell and to a lesser extent, multicellular eukaryotic life. This study compared alpha and beta diversity in eukaryotic communities from seven diverse aquatic environments with pH values ranging from 2 to 11 using massively-parallel pyrotag sequencing targeting the V9 hypervariable region of the 18S ribosomal RNA (rRNA) gene. A total of 946 operational taxonomic units (OTUs) were recovered at a 6% cut-off level (94% similarity) across the sampled environments. Hierarchical clustering of the samples segregated the communities into acidic and alkaline groups. Similarity percentage (SIMPER) analysis followed by indicator OTU analysis (IOA) and non-metric multidimensional scaling (NMDS) were used to determine which characteristic groups of eukaryotic taxa typify acidic or alkaline extremes and the extent to which pH explains eukaryotic community structure in these environments. Spain's Rio Tinto yielded the fewest observed OTUs while Nebraska Sandhills alkaline lakes yielded the most. Distinct OTUs, including metazoan OTUs, numerically dominated pH extreme sites. Indicator OTUs included the diatom Pinnularia and unidentified opisthokonts (Fungi and Filasterea) in the extremely acidic environments, and the ciliate Frontonia across the extremely alkaline sites. Inferred from NMDS, pH explained only a modest fraction of the variation across the datasets, indicating that other factors influence the underlying community structure in these environments. The findings from this study suggest that the ability for eukaryotes to adapt to pH extremes over a broad range of values may be rare, but further study of taxa that can broadly adapt across diverse acidic and alkaline environments, respectively present good models for understanding adaptation and should be targeted for future investigations. PMID:23335919

  12. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

    PubMed Central

    Capone, J P; Sharp, P A; RajBhandary, U L

    1985-01-01

    Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2990894

  13. Construction of eukaryotic expression vector with mBD1-mBD3 fusion genes and exploring its activity against influenza A virus.

    PubMed

    Li, Wanyi; Feng, Yan; Kuang, Yu; Zeng, Wei; Yang, Yuan; Li, Hong; Jiang, Zhonghua; Li, Mingyuan

    2014-01-01

    Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2-6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G₄₁₈ screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID5₅₀ 72 h later. The TCID₅₀ titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID₅₀ titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent

  14. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2010-09-14

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  15. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-10-27

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  16. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-11-17

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  17. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G

    2015-02-03

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  18. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2012-02-14

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  19. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2009-12-01

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  20. Expanding the eukaryotic genetic code

    SciTech Connect

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2013-01-22

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  1. Expanding the eukaryotic genetic code

    DOEpatents

    Chin, Jason W.; Cropp, T. Ashton; Anderson, J. Christopher; Schultz, Peter G.

    2012-05-08

    This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

  2. Analysis of Porphyra Membrane Transporters Demonstrates Gene Transfer among Photosynthetic Eukaryotes and Numerous Sodium-Coupled Transport Systems1[C][W][OA

    PubMed Central

    Chan, Cheong Xin; Zäuner, Simone; Wheeler, Glen; Grossman, Arthur R.; Prochnik, Simon E.; Blouin, Nicolas A.; Zhuang, Yunyun; Benning, Christoph; Berg, Gry Mine; Yarish, Charles; Eriksen, Renée L.; Klein, Anita S.; Lin, Senjie; Levine, Ira; Brawley, Susan H.; Bhattacharya, Debashish

    2012-01-01

    Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters. PMID:22337920

  3. Patterns of Amino Acid Evolution in the Drosophila ananassae Chimeric Gene, siren, Parallel Those of Other Adh-Derived Chimeras

    PubMed Central

    Shih, Hung-Jui; Jones, Corbin D.

    2008-01-01

    siren1 and siren2 are novel alcohol dehydrogenase (Adh)-derived chimeric genes in the Drosophila bipectinata complex. D. ananassae, however, harbors a single homolog of these genes. Like other Adh-derived chimeric genes, siren evolved adaptively shortly after it was formed. These changes likely shifted the catalytic activity of siren. PMID:18780749

  4. Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells.

    PubMed

    Álvarez-Fraga, Laura; Pérez, Astrid; Rumbo-Feal, Soraya; Merino, María; Vallejo, Juan Andrés; Ohneck, Emily J; Edelmann, Richard E; Beceiro, Alejandro; Vázquez-Ucha, Juan C; Valle, Jaione; Actis, Luis A; Bou, Germán; Poza, Margarita

    2016-05-18

    Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii.

  5. Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells

    PubMed Central

    Álvarez-Fraga, Laura; Pérez, Astrid; Rumbo-Feal, Soraya; Merino, María; Vallejo, Juan Andrés; Ohneck, Emily J.; Edelmann, Richard E.; Beceiro, Alejandro; Vázquez-Ucha, Juan C.; Valle, Jaione; Actis, Luis A.; Bou, Germán; Poza, Margarita

    2016-01-01

    ABSTRACT Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii. PMID:26854744

  6. Endosymbiosis and Eukaryotic Cell Evolution.

    PubMed

    Archibald, John M

    2015-10-01

    Understanding the evolution of eukaryotic cellular complexity is one of the grand challenges of modern biology. It has now been firmly established that mitochondria and plastids, the classical membrane-bound organelles of eukaryotic cells, evolved from bacteria by endosymbiosis. In the case of mitochondria, evidence points very clearly to an endosymbiont of α-proteobacterial ancestry. The precise nature of the host cell that partnered with this endosymbiont is, however, very much an open question. And while the host for the cyanobacterial progenitor of the plastid was undoubtedly a fully-fledged eukaryote, how - and how often - plastids moved from one eukaryote to another during algal diversification is vigorously debated. In this article I frame modern views on endosymbiotic theory in a historical context, highlighting the transformative role DNA sequencing played in solving early problems in eukaryotic cell evolution, and posing key unanswered questions emerging from the age of comparative genomics.

  7. Polyethylenimine derivate conjugated with RGD-TAT-NLS as a novel gene vector.

    PubMed

    Zhao, Wenfang; Liu, Kehai; Chen, Shunsheng; Zhu, Man Man; Lv, Hui; Hu, Jing; Mao, Yuan

    2014-01-01

    To solve the contradiction between the cell toxicity and transfection efficiency of polyethylenimine (PEI) derivate in non-viral gene therapy, a novel gene vector, P123-PEI-R18 was synthesized by using biodegradable PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS. The particle size of P123-PEI-R18/DNA was around 100-250 nm. The gene vector could condense DNA at the weight ratio of 2 and protect plasmid DNA from being dissolved in the blood circulation. Importantly, the complexes exhibited lower cell toxicity and higher transfection efficiency contrasted with PEI 25 kDa in vitro. P123-PEI-R18 holds high potential as a safe and efficient gene vector. PMID:25226889

  8. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    PubMed Central

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2014-01-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. PMID:24232694

  9. Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

    PubMed Central

    Müller, Miklós; Mentel, Marek; van Hellemond, Jaap J.; Henze, Katrin; Woehle, Christian; Gould, Sven B.; Yu, Re-Young; van der Giezen, Mark

    2012-01-01

    Summary: Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified. PMID:22688819

  10. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    SciTech Connect

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  11. A New Exon Derived from a Mammalian Apparent LTR Retrotransposon of the SUPT16H Gene

    PubMed Central

    Bae, Min-In; Kim, Yun-Ji; Lee, Ja-Rang; Jung, Yi-Deun; Kim, Heui-Soo

    2013-01-01

    The SUPT16H gene known as FACTP140 is required for the transcription of other genes. For transcription, genes need to be complexed with accessory factors, including transcription factors and RNA polymerase II. One such factor, FACT, interacts with histones H2A/H2B for nucleosome disassembly and transcription elongation. The SUPT16H gene has a transcript and many expressed sequence tags (ESTs). We were especially interested in an MaLR-derived transcript (EST, BX333035) that included a new exon introduced by a transposable element, a mammalian apparent LTR retrotransposon (MaLR). The MaLR was detected ranging from humans to galagos, indicating the MaLR in the SUPT16H gene is integrated into the primate ancestor genome. A new exon was created by alternative donor site provided by the MaLR. The original transcript and the MaLR-derived transcript were expressed in various human, rhesus monkey, and other primate tissues. Additionally, we identified a new alternative transcript that included the MaLR, but there was no significant difference in the expression of the original transcript and the MaLR-derived transcript. Interestingly, the new alternative transcript and the MaLR-derived transcript had the MaLR sequence in the new exon, but they had different structures by adopting different 3′ splice sites. From this study, we verified transposable elements that contributed to transcriptome diversity. PMID:23671841

  12. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

    PubMed

    Boitchenko, Veronika E.; Korobko, Vyacheslav G.; Prassolov, Vladimir S.; Kravchenko, Vladimir V.; Kuimov, Alexander N.; Turetskaya, Regina L.; Kuprash, Dmitry V.; Nedospasov, Sergei A.

    2000-10-01

    Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans. In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha. Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

  13. Multiple Horizontal Gene Transfers of Ammonium Transporters/Ammonia Permeases from Prokaryotes to Eukaryotes: Toward a New Functional and Evolutionary Classification

    PubMed Central

    McDonald, Tami R.; Dietrich, Fred S.; Lutzoni, François

    2012-01-01

    The proteins of the ammonium transporter/methylammonium permease/Rhesus factor family (AMT/MEP/Rh family) are responsible for the movement of ammonia or ammonium ions across the cell membrane. Although it has been established that the Rh proteins are distantly related to the other members of the family, the evolutionary history of the AMT/MEP/Rh family remains unclear. Here, we use phylogenetic analysis to infer the evolutionary history of this family of proteins across 191 genomes representing all main lineages of life and to provide a new classification of the proteins in this family. Our phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). We show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: The AMT family as defined here exhibits vertical gene transfer (i.e., standard parent-to-offspring inheritance), whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs). These ancient HGT events include a gene replacement during the early evolution of the fungi, which could be a defining trait for the kingdom Fungi, a gene gain from hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina) that was subsequently lost in most lineages but retained in even distantly related lichenized fungi. This recircumscription of the ammonium transporters/ammonia permeases family into MEP and AMT families informs the debate on the mechanism of transport in these proteins and on the nature of the transported molecule because published crystal structures of proteins from the MEP and Rh clades may not be representative of the AMT clade. The clades as depicted in this phylogenetic study appear to correspond to

  14. [PCR-derived technology in gene identification and typing of Yersinia pestis].

    PubMed

    Wang, Mei; Tang, Xinyuan; Wang, Zuyun

    2015-01-01

    Application of the PCR-derived technology in gene identification and genotypes of different ecotype Yersinia pestis to make the high-throughput experimental results can reflect the epidemic history and compare the diversity in genome, pathogenicity, so that results from these experiments provide an important basis for clinical diagnosis, treatment and origin. But the experiment should be considered typing ability, practicality, budget and other experimental factors or conditions, because each PCR-derivative technology has advantages and disadvantages.

  15. The evolution of modern eukaryotic phytoplankton.

    PubMed

    Falkowski, Paul G; Katz, Miriam E; Knoll, Andrew H; Quigg, Antonietta; Raven, John A; Schofield, Oscar; Taylor, F J R

    2004-07-16

    The community structure and ecological function of contemporary marine ecosystems are critically dependent on eukaryotic phytoplankton. Although numerically inferior to cyanobacteria, these organisms are responsible for the majority of the flux of organic matter to higher trophic levels and the ocean interior. Photosynthetic eukaryotes evolved more than 1.5 billion years ago in the Proterozoic oceans. However, it was not until the Mesozoic Era (251 to 65 million years ago) that the three principal phytoplankton clades that would come to dominate the modern seas rose to ecological prominence. In contrast to their pioneering predecessors, the dinoflagellates, coccolithophores, and diatoms all contain plastids derived from an ancestral red alga by secondary symbiosis. Here we examine the geological, geochemical, and biological processes that contributed to the rise of these three, distantly related, phytoplankton groups.

  16. Circular RNAs in Eukaryotic Cells.

    PubMed

    Chen, Liang; Huang, Chuan; Wang, Xiaolin; Shan, Ge

    2015-10-01

    Circular RNAs (circRNAs) are now recognized as large species of transcripts in eukaryotic cells. From model organisms such as C. elegans, Drosophila, mice to human beings, thousands of circRNAs formed from back-splicing of exons have been identified. The known complexity of transcriptome has been greatly expanded upon the discovery of these RNAs. Studies about the biogenesis and physiological functions have yielded substantial knowledge for the circRNAs, and they are now more likely to be viewed as regulatory elements coded by the genome rather than unavoidable noise of gene expression. Certain human diseases may also relate to circRNAs. These circRNAs show diversifications in features such as sequence composition and cellular localization, and thus we propose that they may be divided into subtypes such as cytoplasmic circRNAs, nuclear circRNAs, and exon-intron circRNAs (EIciRNAs). Here we summarize and discuss knowns and unknowns for these RNAs, and we need to keep in mind that the whole field is still at the beginning of exciting explorations.

  17. Minimal genomes of mycoplasma-related endobacteria are plastic and contain host-derived genes for sustained life within Glomeromycota.

    PubMed

    Naito, Mizue; Morton, Joseph B; Pawlowska, Teresa E

    2015-06-23

    Arbuscular mycorrhizal fungi (AMF, Glomeromycota) colonize roots of the majority of terrestrial plants. They provide essential minerals to their plant hosts and receive photosynthates in return. All major lineages of AMF harbor endobacteria classified as Mollicutes, and known as mycoplasma-related endobacteria (MRE). Except for their substantial intrahost genetic diversity and ability to transmit vertically, virtually nothing is known about the life history of these endobacteria. To understand MRE biology, we sequenced metagenomes of three MRE populations, each associated with divergent AMF hosts. We found that each AMF species harbored a genetically distinct group of MRE. Despite vertical transmission, all MRE populations showed extensive chromosomal rearrangements, which we attributed to genetic recombination, activity of mobile elements, and a history of plectroviral invasion. The MRE genomes are characterized by a highly reduced gene content, indicating metabolic dependence on the fungal host, with the mechanism of energy production remaining unclear. Several MRE genes encode proteins with domains involved in protein-protein interactions with eukaryotic hosts. In addition, the MRE genomes harbor genes horizontally acquired from AMF. Some of these genes encode small ubiquitin-like modifier (SUMO) proteases specific to the SUMOylation systems of eukaryotes, which MRE likely use to manipulate their fungal host. The extent of MRE genome plasticity and reduction, along with the large number of horizontally acquired host genes, suggests a high degree of adaptation to the fungal host. These features, together with the ubiquity of the MRE-Glomeromycota associations, emphasize the significance of MRE in the biology of Glomeromycota.

  18. Synchronization of Eukaryotic Flagella

    NASA Astrophysics Data System (ADS)

    Goldstein, Raymond E.

    2012-11-01

    From unicellular organisms as small as a few microns to the largest vertebrates on earth we find groups of beating flagella or cilia that exhibit striking spatio-temporal organization. This may take the form of precise frequency and phase locking as frequently found in the swimming of green algae, or beating with long-wavelength phase modulations known as metachronal waves, seen in ciliates and in our respiratory systems. The remarkable similarity in the underlying molecular structure of flagella across the whole eukaryotic world leads naturally to the hypothesis that a similarly universal mechanism might be responsible for synchronization. Although this mechanism is poorly understood, one appealing hypothesis is that it results from hydrodynamic interactions between flagella. In this talk I will describe a synthesis of recent experimental and theoretical studies of this issue that have provided the strongest evidence to date for the hydrodynamic origin of flagellar synchronization. At the unicellular level this includes studies of the beating of the two flagella of the wild type unicellular alga Chlamydomonas reinhardtii in their native state and under conditions of regrowth following autotomy, and of the flagellar dominance mutant ptx1, which displays unusual anti-phase synchronization. Analysis of the related multicellular organism Volvox carteri shows it to be an ideal model organism for the study of metachronal waves. Supported by BBSRC, EPSRC, ERC, and The Wellcome Trust.

  19. Transcriptome-Derived Tetranucleotide Microsatellites and Their Associated Genes from the Giant Panda (Ailuropoda melanoleuca).

    PubMed

    Song, Xuhao; Shen, Fujun; Huang, Jie; Huang, Yan; Du, Lianming; Wang, Chengdong; Fan, Zhenxin; Hou, Rong; Yue, Bisong; Zhang, Xiuyue

    2016-09-01

    Recently, an increasing number of microsatellites or simple sequence repeats (SSRs) have been found and characterized from transcriptomes. Such SSRs can be employed as putative functional markers to easily tag corresponding genes, which play an important role in biomedical studies and genetic analysis. However, the transcriptome-derived SSRs for giant panda (Ailuropoda melanoleuca) are not yet available. In this work, we identified and characterized 20 tetranucleotide microsatellite loci from a transcript database generated from the blood of giant panda. Furthermore, we assigned their predicted transcriptome locations: 16 loci were assigned to untranslated regions (UTRs) and 4 loci were assigned to coding regions (CDSs). Gene identities of 14 transcripts contained corresponding microsatellites were determined, which provide useful information to study the potential contribution of SSRs to gene regulation in giant panda. The polymorphic information content (PIC) values ranged from 0.293 to 0.789 with an average of 0.603 for the 16 UTRs-derived SSRs. Interestingly, 4 CDS-derived microsatellites developed in our study were also polymorphic, and the instability of these 4 CDS-derived SSRs was further validated by re-genotyping and sequencing. The genes containing these 4 CDS-derived SSRs were embedded with various types of repeat motifs. The interaction of all the length-changing SSRs might provide a way against coding region frameshift caused by microsatellite instability. We hope these newly gene-associated biomarkers will pave the way for genetic and biomedical studies for giant panda in the future. In sum, this set of transcriptome-derived markers complements the genetic resources available for giant panda.

  20. Transcriptome-Derived Tetranucleotide Microsatellites and Their Associated Genes from the Giant Panda (Ailuropoda melanoleuca).

    PubMed

    Song, Xuhao; Shen, Fujun; Huang, Jie; Huang, Yan; Du, Lianming; Wang, Chengdong; Fan, Zhenxin; Hou, Rong; Yue, Bisong; Zhang, Xiuyue

    2016-09-01

    Recently, an increasing number of microsatellites or simple sequence repeats (SSRs) have been found and characterized from transcriptomes. Such SSRs can be employed as putative functional markers to easily tag corresponding genes, which play an important role in biomedical studies and genetic analysis. However, the transcriptome-derived SSRs for giant panda (Ailuropoda melanoleuca) are not yet available. In this work, we identified and characterized 20 tetranucleotide microsatellite loci from a transcript database generated from the blood of giant panda. Furthermore, we assigned their predicted transcriptome locations: 16 loci were assigned to untranslated regions (UTRs) and 4 loci were assigned to coding regions (CDSs). Gene identities of 14 transcripts contained corresponding microsatellites were determined, which provide useful information to study the potential contribution of SSRs to gene regulation in giant panda. The polymorphic information content (PIC) values ranged from 0.293 to 0.789 with an average of 0.603 for the 16 UTRs-derived SSRs. Interestingly, 4 CDS-derived microsatellites developed in our study were also polymorphic, and the instability of these 4 CDS-derived SSRs was further validated by re-genotyping and sequencing. The genes containing these 4 CDS-derived SSRs were embedded with various types of repeat motifs. The interaction of all the length-changing SSRs might provide a way against coding region frameshift caused by microsatellite instability. We hope these newly gene-associated biomarkers will pave the way for genetic and biomedical studies for giant panda in the future. In sum, this set of transcriptome-derived markers complements the genetic resources available for giant panda. PMID:27112165

  1. Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts

    PubMed Central

    Bhadury, Joydeep; Einarsdottir, Berglind O.; Podraza, Agnieszka; Bagge, Roger Olofsson; Stierner, Ulrika; Ny, Lars; López, Marcela Dávila; Nilsson, Jonas A.

    2016-01-01

    Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors. PMID:27009863

  2. Archaeal ancestors of eukaryotes: not so elusive any more.

    PubMed

    Koonin, Eugene V

    2015-01-01

    The origin of eukaryotes is one of the hardest problems in evolutionary biology and sometimes raises the ominous specter of irreducible complexity. Reconstruction of the gene repertoire of the last eukaryotic common ancestor (LECA) has revealed a highly complex organism with a variety of advanced features but no detectable evolutionary intermediates to explain their origin. Recently, however, genome analysis of diverse archaea led to the discovery of apparent ancestral versions of several signature eukaryotic systems, such as the actin cytoskeleton and the ubiquitin network, that are scattered among archaea. These findings inspired the hypothesis that the archaeal ancestor of eukaryotes was an unusually complex form with an elaborate intracellular organization. The latest striking discovery made by deep metagenomic sequencing vindicates this hypothesis by showing that in phylogenetic trees eukaryotes fall within a newly identified archaeal group, the Lokiarchaeota, which combine several eukaryotic signatures previously identified in different archaea. The discovery of complex archaea that are the closest living relatives of eukaryotes is most compatible with the symbiogenetic scenario for eukaryogenesis.

  3. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

    PubMed

    Wijffels, René H; Kruse, Olaf; Hellingwerf, Klaas J

    2013-06-01

    Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments. Cyanobacteria are promising host organisms for the production of small molecules that can be secreted such as ethanol, butanol, fatty acids and other organic acids. Eukaryotic microalgae are interesting for products for which cellular storage is important such as proteins, lipids, starch and alkanes. For the development of new and promising lines of production, strains of both cyanobacteria and eukaryotic microalgae have to be improved. Transformation systems have been much better developed in cyanobacteria. However, several products would be preferably produced with eukaryotic microalgae. In the case of cyanobacteria a synthetic-systems biology approach has a great potential to exploit cyanobacteria as cell factories. For eukaryotic microalgae transformation systems need to be further developed. A promising strategy is transformation of heterologous (prokaryotic and eukaryotic) genes in established eukaryotic hosts such as Chlamydomonas reinhardtii. Experimental outdoor pilots under containment for the production of genetically modified cyanobacteria and microalgae are in progress. For full scale production risks of release of genetically modified organisms need to be assessed.

  4. Characterization of a Soil Metagenome-Derived Gene Encoding Wax Ester Synthase.

    PubMed

    Kim, Nam Hee; Park, Ji-Hye; Chung, Eunsook; So, Hyun-Ah; Lee, Myung Hwan; Kim, Jin-Cheol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-02-01

    A soil metagenome contains the genomes of all microbes included in a soil sample, including those that cannot be cultured. In this study, soil metagenome libraries were searched for microbial genes exhibiting lipolytic activity and those involved in potential lipid metabolism that could yield valuable products in microorganisms. One of the subclones derived from the original fosmid clone, pELP120, was selected for further analysis. A subclone spanning a 3.3 kb DNA fragment was found to encode for lipase/esterase and contained an additional partial open reading frame encoding a wax ester synthase (WES) motif. Consequently, both pELP120 and the full length of the gene potentially encoding WES were sequenced. To determine if the wes gene encoded a functioning WES protein that produced wax esters, gas chromatography-mass spectroscopy was conducted using ethyl acetate extract from an Escherichia coli strain that expressed the wes gene and was grown with hexadecanol. The ethyl acetate extract from this E. coli strain did indeed produce wax ester compounds of various carbon-chain lengths. DNA sequence analysis of the full-length gene revealed that the gene cluster may be derived from a member of Proteobacteria, whereas the clone does not contain any clear phylogenetic markers. These results suggest that the wes gene discovered in this study encodes a functional protein in E. coli and produces wax esters through a heterologous expression system.

  5. A Eukaryotic-Acquired Gene by a Biotrophic Phytopathogen Allows Prolonged Survival on the Host by Counteracting the Shut-Down of Plant Photosynthesis

    PubMed Central

    Gottig, Natalia; Dunger, Germán; Garofalo, Cecilia G.; Daurelio, Lucas D.; Ndimba, Bongani; Orellano, Elena G.; Gehring, Chris; Ottado, Jorgelina

    2010-01-01

    Xanthomonas citri pv. citri, the bacteria responsible for citrus canker posses a biological active plant natriuretic peptide (PNP)-like protein, not present in any other bacteria. PNPs are a class of extracellular, systemically mobile peptides that elicit a number of plant responses important in homeostasis and growth. Previously, we showed that a Xanthomonas citri pv. citri mutant lacking the PNP-like protein XacPNP produced more necrotic lesions in citrus leaves than wild type infections and suggested a role for XacPNP in the regulation of host homeostasis. Here we have analyzed the proteome modifications observed in citrus leaves infected with the wild type and XacPNP deletion mutant bacteria. While both of them cause down-regulation of enzymes related to photosynthesis as well as chloroplastic ribosomal proteins, proteins related to defense responses are up-regulated. However, leaves infiltrated with the XacPNP deletion mutant show a more pronounced decrease in photosynthetic proteins while no reduction in defense related proteins as compared to the wild-type pathogen. This suggests that XacPNP serves the pathogen to maintain host photosynthetic efficiency during pathogenesis. The results from the proteomics analyses are consistent with our chlorophyll fluorescence data and transcript analyses of defense genes that show a more marked reduction in photosynthesis in the mutant but no difference in the induction of genes diagnostic for biotic-stress responses. We therefore conclude that XacPNP counteracts the shut-down of host photosynthesis during infection and in that way maintains the tissue in better conditions, suggesting that the pathogen has adapted a host gene to modify its natural host and render it a better reservoir for prolonged bacterial survival and thus for further colonization. PMID:20126632

  6. Translational control of intron splicing in eukaryotes.

    PubMed

    Jaillon, Olivier; Bouhouche, Khaled; Gout, Jean-François; Aury, Jean-Marc; Noel, Benjamin; Saudemont, Baptiste; Nowacki, Mariusz; Serrano, Vincent; Porcel, Betina M; Ségurens, Béatrice; Le Mouël, Anne; Lepère, Gersende; Schächter, Vincent; Bétermier, Mireille; Cohen, Jean; Wincker, Patrick; Sperling, Linda; Duret, Laurent; Meyer, Eric

    2008-01-17

    Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

  7. Gene mutations in primary tumors and corresponding patient-derived xenografts derived from non-small cell lung cancer

    PubMed Central

    Peng, Shaohua; Cao, Mengru; Li, Hongyu; Hu, Jing; Huang, Xiao; Liu, Wei; Zhang, Hui; Wu, Shuhong; Pataer, Apar; Heymach, John V.; Eterovic, Agda Karina; Zhang, Qingxiu; Shaw, Kenna R.; Chen, Ken; Futreal, Andrew; Wang, Michael; Hofstetter, Wayne; Mehran, Reza; Rice, David; Roth, Jack A.; Sepesi, Boris; Swisher, Stephen G.; Vaporciyan, Ara; Walsh, Garrett L.; Johnson, Faye M.; Fang, Bingliang

    2014-01-01

    Molecular annotated patient-derived xenograft (PDX) models are useful for the preclinical investigation of anticancer drugs and individualized anticancer therapy. We established 23 PDXs from 88 surgical specimens of lung cancer patients and determined gene mutations in these PDXs and their paired primary tumors by ultradeep exome sequencing on 202 cancer-related genes. The numbers of primary tumors with deleterious mutations in TP53, KRAS, PI3KCA, ALK, STK11, and EGFR were 43.5%, 21.7%, 17.4%, 17.4%, 13.0%, and 8.7%, respectively. Other genes with deleterious mutations in ≥3 (13.0%) primary tumors were MLL3, SETD2, ATM, ARID1A, CRIPAK, HGF, BAI3, EP300, KDR, PDGRRA and RUNX1. Of 315 mutations detected in the primary tumors, 293 (93%) were also detected in their corresponding PDXs, indicating that PDXs have the capacity to recapitulate the mutations in primary tumors. Nevertheless, a substantial number of mutations had higher allele frequencies in the PDXs than in the primary tumors, or were not detectable in the primary tumor, suggesting the possibility of tumor cell enrichment in PDXs or heterogeneity in the primary tumors. The molecularly annotated PDXs generated from this study could be useful for future translational studies. PMID:25444907

  8. Gene mutations in primary tumors and corresponding patient-derived xenografts derived from non-small cell lung cancer.

    PubMed

    Hao, Chuncheng; Wang, Li; Peng, Shaohua; Cao, Mengru; Li, Hongyu; Hu, Jing; Huang, Xiao; Liu, Wei; Zhang, Hui; Wu, Shuhong; Pataer, Apar; Heymach, John V; Eterovic, Agda Karina; Zhang, Qingxiu; Shaw, Kenna R; Chen, Ken; Futreal, Andrew; Wang, Michael; Hofstetter, Wayne; Mehran, Reza; Rice, David; Roth, Jack A; Sepesi, Boris; Swisher, Stephen G; Vaporciyan, Ara; Walsh, Garrett L; Johnson, Faye M; Fang, Bingliang

    2015-02-01

    Molecular annotated patient-derived xenograft (PDX) models are useful for the preclinical investigation of anticancer drugs and individualized anticancer therapy. We established 23 PDXs from 88 surgical specimens of lung cancer patients and determined gene mutations in these PDXs and their paired primary tumors by ultradeep exome sequencing on 202 cancer-related genes. The numbers of primary tumors with deleterious mutations in TP53, KRAS, PI3KCA, ALK, STK11, and EGFR were 43.5%, 21.7%, 17.4%, 17.4%, 13.0%, and 8.7%, respectively. Other genes with deleterious mutations in ≥3 (13.0%) primary tumors were MLL3, SETD2, ATM, ARID1A, CRIPAK, HGF, BAI3, EP300, KDR, PDGRRA and RUNX1. Of 315 mutations detected in the primary tumors, 293 (93%) were also detected in their corresponding PDXs, indicating that PDXs have the capacity to recapitulate the mutations in primary tumors. Nevertheless, a substantial number of mutations had higher allele frequencies in the PDXs than in the primary tumors, or were not detectable in the primary tumor, suggesting the possibility of tumor cell enrichment in PDXs or heterogeneity in the primary tumors. The molecularly annotated PDXs generated from this study could be useful for future translational studies.

  9. NADPH oxidases in Eukaryotes: red algae provide new hints!

    PubMed

    Hervé, Cécile; Tonon, Thierry; Collén, Jonas; Corre, Erwan; Boyen, Catherine

    2006-03-01

    The red macro-alga Chondrus crispus is known to produce superoxide radicals in response to cell-free extracts of its green algal pathogenic endophyte Acrochaete operculata. So far, no enzymes involved in this metabolism have been isolated from red algae. We report here the isolation of a gene encoding a homologue of the respiratory burst oxidase gp91(phox) in C. crispus, named Ccrboh. This single copy gene encodes a polypeptide of 825 amino acids. Search performed in available genome and EST algal databases identified sequences showing common features of NADPH oxidases in other algae such as the red unicellular Cyanidioschyzon merolae, the economically valuable red macro-alga Porphyra yezoensis and the two diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Domain organization and phylogenetic relationships with plant, animal, fungal and algal NADPH oxidase homologues were analyzed. Transcription analysis of the C. crispus gene revealed that it was over-transcribed during infection of C. crispus gametophyte by the endophyte A. operculata, and after incubation in presence of atrazine, methyl jasmonate and hydroxyperoxides derived from C20 polyunsaturated fatty acids (PUFAs). These results also illustrate the interest of exploring the red algal lineage for gaining insight into the deep evolution of NADPH oxidases in Eukaryotes.

  10. Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

    PubMed

    Osińska, Magdalena; Wiejak, Jolanta; Wypych, Emilia; Bilski, Henryk; Bartosiewicz, Rafał; Wyroba, Elżbieta

    2011-01-01

    Rab7 GTPases are involved in membrane trafficking in the late endosomal/lysosomal pathway. In Paramecium octaurelia Rab7a and Rab7b are encoded by paralogous genes. Antipeptide antibodies generated against divergent C-termini recognize Rab7a of 22.5 kDa and Rab7b of 25 kDa, respectively. In 2D gel electrophoresis two immunoreactive spots were identified for Rab7b at pI about 6.34 and about 6.18 and only one spot for Rab7a of pI about 6.34 suggesting post-translational modification of Rab7b. Mass spectrometry revealed eight identical phosphorylated residues in the both proteins. ProQ Emerald staining and ConA overlay of immunoprecipitated Rab7b indicated its putative glycosylation that was further supported by a faster electrophoretic mobility of this protein upon deglycosylation. Such a post-translational modification and substitution of Ala(140) in Rab7a for Ser(140) in Rab7b may result in distinct targeting to the oral apparatus where Rab7b associates with the microtubular structures as revealed by STED confocal and electron microscopy. Rab7a was mapped to phagosomal compartment. Absolute qReal-Time PCR analysis revealed that expression of Rab7a was 2.6-fold higher than that of Rab7b. Upon latex internalization it was further 2-fold increased for Rab7a and only slightly for Rab7b. Post-transcriptional gene silencing of rab7a suppressed phagosome formation by 70 % and impaired their acidification. Ultrastructural analysis with double immunogold labeling revealed that this effect was due to the lack of V-ATPase recruitment to phagolysosomes. No significant phenotype changes were noticed in cells upon rab7b silencing. In conclusion, Rab7b acquired a new function, whereas Rab7a can be assigned to the phagolysosomal pathway.

  11. Diurnal Transcriptional Regulation of Endosymbiotically Derived Genes in the Chlorarachniophyte Bigelowiella natans.

    PubMed

    Suzuki, Shigekatsu; Ishida, Ken-Ichiro; Hirakawa, Yoshihisa

    2016-01-01

    Chlorarachniophyte algae possess complex plastids acquired by the secondary endosymbiosis of a green alga, and the plastids harbor a relict nucleus of the endosymbiont, the so-called nucleomorph. Due to massive gene transfer from the endosymbiont to the host, many proteins involved in plastid and nucleomorph are encoded by the nuclear genome. Genome sequences have provided a blueprint for the fate of endosymbiotically derived genes; however, transcriptional regulation of these genes remains poorly understood. To gain insight into the evolution of endosymbiotic genes, we performed genome-wide transcript profiling along the cell cycle of the chlorarachniophyte Bigelowiella natans, synchronized by light and dark cycles. Our comparative analyses demonstrated that transcript levels of 7,751 nuclear genes (35.7% of 21,706 genes) significantly oscillated along the diurnal/cell cycles, and those included 780 and 147 genes for putative plastid and nucleomorph-targeted proteins, respectively. Clustering analysis of those genes revealed the existence of transcriptional networks related to specific biological processes such as photosynthesis, carbon metabolism, translation, and DNA replication. Interestingly, transcripts of many plastid-targeted proteins in B. natans were induced before dawn, unlike other photosynthetic organisms. In contrast to nuclear genes, 99% nucleomorph genes were found to be constitutively expressed during the cycles. We also found that the nucleomorph DNA replication would be controlled by a nucleus-encoded viral-like DNA polymerase. The results of this study suggest that nucleomorph genes have lost transcriptional regulation along the diurnal cycles, and nuclear genes exert control over the complex plastid including the nucleomorph. PMID:27503292

  12. A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms.

    PubMed

    Torrens-Spence, M P; Fallon, T R; Weng, J K

    2016-01-01

    Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. PMID:27480683

  13. The Eukaryotic Tree of Life from a Global Phylogenomic Perspective

    PubMed Central

    Burki, Fabien

    2014-01-01

    Molecular phylogenetics has revolutionized our knowledge of the eukaryotic tree of life. With the advent of genomics, a new discipline of phylogenetics has emerged: phylogenomics. This method uses large alignments of tens to hundreds of genes to reconstruct evolutionary histories. This approach has led to the resolution of ancient and contentious relationships, notably between the building blocks of the tree (the supergroups), and allowed to place in the tree enigmatic yet important protist lineages for understanding eukaryote evolution. Here, I discuss the pros and cons of phylogenomics and review the eukaryotic supergroups in light of earlier work that laid the foundation for the current view of the tree, including the position of the root. I conclude by presenting a picture of eukaryote evolution, summarizing the most recent progress in assembling the global tree. PMID:24789819

  14. An ancestral bacterial division system is widespread in eukaryotic mitochondria.

    PubMed

    Leger, Michelle M; Petrů, Markéta; Žárský, Vojtěch; Eme, Laura; Vlček, Čestmír; Harding, Tommy; Lang, B Franz; Eliáš, Marek; Doležal, Pavel; Roger, Andrew J

    2015-08-18

    Bacterial division initiates at the site of a contractile Z-ring composed of polymerized FtsZ. The location of the Z-ring in the cell is controlled by a system of three mutually antagonistic proteins, MinC, MinD, and MinE. Plastid division is also known to be dependent on homologs of these proteins, derived from the ancestral cyanobacterial endosymbiont that gave rise to plastids. In contrast, the mitochondria of model systems such as Saccharomyces cerevisiae, mammals, and Arabidopsis thaliana seem to have replaced the ancestral α-proteobacterial Min-based division machinery with host-derived dynamin-related proteins that form outer contractile rings. Here, we show that the mitochondrial division system of these model organisms is the exception, rather than the rule, for eukaryotes. We describe endosymbiont-derived, bacterial-like division systems comprising FtsZ and Min proteins in diverse less-studied eukaryote protistan lineages, including jakobid and heterolobosean excavates, a malawimonad, stramenopiles, amoebozoans, a breviate, and an apusomonad. For two of these taxa, the amoebozoan Dictyostelium purpureum and the jakobid Andalucia incarcerata, we confirm a mitochondrial localization of these proteins by their heterologous expression in Saccharomyces cerevisiae. The discovery of a proteobacterial-like division system in mitochondria of diverse eukaryotic lineages suggests that it was the ancestral feature of all eukaryotic mitochondria and has been supplanted by a host-derived system multiple times in distinct eukaryote lineages.

  15. Mitochondrial genome evolution and the origin of eukaryotes.

    PubMed

    Lang, B F; Gray, M W; Burger, G

    1999-01-01

    Recent results from ancestral (minimally derived) protists testify to the tremendous diversity of the mitochondrial genome in various eukaryotic lineages, but also reinforce the view that mitochondria, descendants of an endosymbiotic alpha-Proteobacterium, arose only once in evolution. The serial endosymbiosis theory, currently the most popular hypothesis to explain the origin of mitochondria, postulates the capture of an alpha-proteobacterial endosymbiont by a nucleus-containing eukaryotic host resembling extant amitochondriate protists. New sequence data have challenged this scenario, instead raising the possibility that the origin of the mitochondrion was coincident with, and contributed substantially to, the origin of the nuclear genome of the eukaryotic cell. Defining more precisely the alpha-proteobacterial ancestry of the mitochondrial genome, and the contribution of the endosymbiotic event to the nuclear genome, will be essential for a full understanding of the origin and evolution of the eukaryotic cell as a whole.

  16. Coding sequences of functioning human genes derived entirely from mobile element sequences.

    PubMed

    Britten, Roy J

    2004-11-30

    Among all of the many examples of mobile elements or "parasitic sequences" that affect the function of the human genome, this paper describes several examples of functioning genes whose sequences have been almost completely derived from mobile elements. There are many examples where the synthetic coding sequences of observed mRNA sequences are made up of mobile element sequences, to an extent of 80% or more of the length of the coding sequences. In the examples described here, the genes have named functions, and some of these functions have been studied. It appears that each of the functioning genes was originally formed from mobile elements and that in some process of molecular evolution a coding sequence was derived that could be translated into a protein that is of some importance to human biology. In one case (AD7C), the coding sequence is 99% made up of a cluster of Alu sequences. In another example, the gene BNIP3 coding sequence is 97% made up of sequences from an apparent human endogenous retrovirus. The Syncytin gene coding sequence appears to be made from an endogenous retrovirus envelope gene. PMID:15546984

  17. Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

    PubMed

    Jones, Adam C; Gust, Bertolt; Kulik, Andreas; Heide, Lutz; Buttner, Mark J; Bibb, Mervyn J

    2013-01-01

    We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

  18. An archaeal origin of eukaryotes supports only two primary domains of life.

    PubMed

    Williams, Tom A; Foster, Peter G; Cox, Cymon J; Embley, T Martin

    2013-12-12

    The discovery of the Archaea and the proposal of the three-domains 'universal' tree, based on ribosomal RNA and core genes mainly involved in protein translation, catalysed new ideas for cellular evolution and eukaryotic origins. However, accumulating evidence suggests that the three-domains tree may be incorrect: evolutionary trees made using newer methods place eukaryotic core genes within the Archaea, supporting hypotheses in which an archaeon participated in eukaryotic origins by founding the host lineage for the mitochondrial endosymbiont. These results provide support for only two primary domains of life--Archaea and Bacteria--because eukaryotes arose through partnership between them.

  19. Origins of Eukaryotic Sexual Reproduction

    PubMed Central

    2014-01-01

    Sexual reproduction is a nearly universal feature of eukaryotic organisms. Given its ubiquity and shared core features, sex is thought to have arisen once in the last common ancestor to all eukaryotes. Using the perspectives of molecular genetics and cell biology, we consider documented and hypothetical scenarios for the instantiation and evolution of meiosis, fertilization, sex determination, uniparental inheritance of organelle genomes, and speciation. PMID:24591519

  20. Soil eukaryotic functional diversity, a metatranscriptomic approach.

    PubMed

    Bailly, Julie; Fraissinet-Tachet, Laurence; Verner, Marie-Christine; Debaud, Jean-Claude; Lemaire, Marc; Wésolowski-Louvel, Micheline; Marmeisse, Roland

    2007-11-01

    To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

  1. Comparison of eukaryotic phytobenthic community composition in a polluted river by partial 18S rRNA gene cloning and sequencing.

    PubMed

    Dorigo, U; Bérard, A; Humbert, J F

    2002-11-01

    We compared the species composition in phytobenthic communities at different sampling sites in a small French river presenting polluted and unpolluted areas. For each sampling point, the total DNA was extracted and used to construct an 18S rRNA gene clone library after PCR amplification of a ca 400 bp fragment. Phytobenthic community composition was estimated by random sequencing of several clones per library. Most of the sequences corresponded to the Bacillariophyceae and Chlorophyceae groups. By combining phylogenetic and correspondence analyses, we showed that our molecular approach is able to estimate and compare the species composition at different sampling sites in order to assess the environmental impact of xenobiotics on phytobenthic communities. Changes in species composition of these communities were found, but no evident decrease in the diversity. We discuss the significance of these changes with regard to the existing level of pollution and their impact on the functionality of the ecosystem. Our findings suggest that it is now possible to use faster molecular methods (DGGE, ARISA.) to test large numbers of samples in the context of ecotoxicological studies, and thus to assess the impact of pollution in an aquatic ecosystem.

  2. Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

    NASA Astrophysics Data System (ADS)

    Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

    2011-09-01

    In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

  3. Insights into corn genes derived from large-scale cDNA sequencing.

    PubMed

    Alexandrov, Nickolai N; Brover, Vyacheslav V; Freidin, Stanislav; Troukhan, Maxim E; Tatarinova, Tatiana V; Zhang, Hongyu; Swaller, Timothy J; Lu, Yu-Ping; Bouck, John; Flavell, Richard B; Feldmann, Kenneth A

    2009-01-01

    We present a large portion of the transcriptome of Zea mays, including ESTs representing 484,032 cDNA clones from 53 libraries and 36,565 fully sequenced cDNA clones, out of which 31,552 clones are non-redundant. These and other previously sequenced transcripts have been aligned with available genome sequences and have provided new insights into the characteristics of gene structures and promoters within this major crop species. We found that although the average number of introns per gene is about the same in corn and Arabidopsis, corn genes have more alternatively spliced isoforms. Examination of the nucleotide composition of coding regions reveals that corn genes, as well as genes of other Poaceae (Grass family), can be divided into two classes according to the GC content at the third position in the amino acid encoding codons. Many of the transcripts that have lower GC content at the third position have dicot homologs but the high GC content transcripts tend to be more specific to the grasses. The high GC content class is also enriched with intronless genes. Together this suggests that an identifiable class of genes in plants is associated with the Poaceae divergence. Furthermore, because many of these genes appear to be derived from ancestral genes that do not contain introns, this evolutionary divergence may be the result of horizontal gene transfer from species not only with different codon usage but possibly that did not have introns, perhaps outside of the plant kingdom. By comparing the cDNAs described herein with the non-redundant set of corn mRNAs in GenBank, we estimate that there are about 50,000 different protein coding genes in Zea. All of the sequence data from this study have been submitted to DDBJ/GenBank/EMBL under accession numbers EU940701-EU977132 (FLI cDNA) and FK944382-FL482108 (EST). PMID:18937034

  4. Helitrons on a roll: eukaryotic rolling-circle transposons.

    PubMed

    Kapitonov, Vladimir V; Jurka, Jerzy

    2007-10-01

    Rolling-circle eukaryotic transposons, known as Helitron transposons, were first discovered in plants (Arabidopsis thaliana and Oryza sativa) and in the nematode Caenorhabditis elegans. To date, Helitrons have been identified in a diverse range of species, from protists to mammals. They represent a major class of eukaryotic transposons and are fundamentally different from classical transposons in terms of their structure and mechanism of transposition. Helitrons seem to have a major role in the evolution of host genomes. They frequently capture diverse host genes, some of which can evolve into novel host genes or become essential for helitron transposition.

  5. Expression of eukaryotic polypeptides in chloroplasts

    DOEpatents

    Mayfield, Stephen P.

    2013-06-04

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  6. Biosafety of gene therapy vectors derived from herpes simplex virus type 1.

    PubMed

    Lim, Filip; Khalique, Hena; Ventosa, Maria; Baldo, Aline

    2013-12-01

    The majority of humans have been infected with Herpes Simplex Virus Type 1 (HSV-1) and harbor its viral DNA in the latent form within neurons for lifetime. This, combined with the absence of serious adverse effects due to HSV-1 derived vectors in clinical trials so far, highlight the potential to use this virus to develop neuronal gene transfer vectors which are transparent to the host, allowing the effects of the transgene to act without interference from the transfer system eg., for functional genomics in basic neuroscience or gene therapy of neurological disorders. On the other hand, other HSV-1 derived vectors which also have a promising perspective in the clinic, are designed to have enhanced cytotoxicity in certain cell types, as in the case of oncolytic vectors. Understanding virus-host interactions is fundamental not only to the success of these gene therapy vectors but also with respect to identifying and minimizing biohazards associated with their use. In this review we discuss characteristics of HSV-1 and gene therapy vectors derived from this virus which are useful to consider in the context of biosafety risk assessment and risk management.

  7. Archaeal "dark matter" and the origin of eukaryotes.

    PubMed

    Williams, Tom A; Embley, T Martin

    2014-03-01

    Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis.

  8. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    NASA Astrophysics Data System (ADS)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  9. General considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination.

    PubMed

    Baldo, Aline; van den Akker, Eric; Bergmans, Hans E; Lim, Filip; Pauwels, Katia

    2013-12-01

    This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used in human gene therapy and/or vaccination. The importance to assess the potential risks for human health and the environment related to the use of genetically modified organisms (GMO) in this case genetically modified viral vectors is highlighted by several examples. This environmental risk assessment is one of the requirements within the European regulatory framework covering the conduct of clinical trials using GMO. Risk assessment methodologies for the environmental risk assessment of genetically modified virus-derived vectors have been developed. PMID:24195604

  10. General considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination.

    PubMed

    Baldo, Aline; van den Akker, Eric; Bergmans, Hans E; Lim, Filip; Pauwels, Katia

    2013-12-01

    This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used in human gene therapy and/or vaccination. The importance to assess the potential risks for human health and the environment related to the use of genetically modified organisms (GMO) in this case genetically modified viral vectors is highlighted by several examples. This environmental risk assessment is one of the requirements within the European regulatory framework covering the conduct of clinical trials using GMO. Risk assessment methodologies for the environmental risk assessment of genetically modified virus-derived vectors have been developed.

  11. General Considerations on the Biosafety of Virus-derived Vectors Used in Gene Therapy and Vaccination

    PubMed Central

    Baldo, Aline; van den Akker, Eric; Bergmans, Hans E.; Lim, Filip; Pauwels, Katia

    2013-01-01

    This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used in human gene therapy and/or vaccination. The importance to assess the potential risks for human health and the environment related to the use of genetically modified organisms (GMO) in this case genetically modified viral vectors is highlighted by several examples. This environmental risk assessment is one of the requirements within the European regulatory framework covering the conduct of clinical trials using GMO. Risk assessment methodologies for the environmental risk assessment of genetically modified virus-derived vectors have been developed. PMID:24195604

  12. Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity.

    PubMed

    Moon-van der Staay, S Y; De Wachter, R; Vaulot, D

    2001-02-01

    Picoplankton--cells with a diameter of less than 3 microm--are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.

  13. Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity

    NASA Astrophysics Data System (ADS)

    Moon-van der Staay, Seung Yeo; De WachterRDanielVaulot, RupertDe WachterR.Daniel

    2001-02-01

    Picoplankton-cells with a diameter of less than 3µm-are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.

  14. Phylogenomics of Sterol Synthesis: Insights into the Origin, Evolution, and Diversity of a Key Eukaryotic Feature

    PubMed Central

    Desmond, Elie

    2009-01-01

    The availability of complete genomes from a wide sampling of eukaryotic diversity has allowed the application of phylogenomics approaches to study the origin and evolution of unique eukaryotic cellular structures, but these are still poorly applied to study unique eukaryotic metabolic pathways. Sterols are a good example because they are an essential feature of eukaryotic membranes. The sterol pathway has been well dissected in vertebrates, fungi, and land plants. However, although different types of sterols have been identified in other eukaryotic lineages, their pathways have not been fully characterized. We have carried out an extensive analysis of the taxonomic distribution and phylogeny of the enzymes of the sterol pathway in a large sampling of eukaryotic lineages. This allowed us to tentatively indicate features of the sterol pathway in organisms where this has not been characterized and to point out a number of steps for which yet-to-discover enzymes may be at work. We also inferred that the last eukaryotic common ancestor already harbored a large panel of enzymes for sterol synthesis and that subsequent evolution over the eukaryotic tree occurred by tinkering, mainly by gene losses. We highlight a high capacity of sterol synthesis in the myxobacterium Plesiocystis pacifica, and we support the hypothesis that the few bacteria that harbor homologs of the sterol pathway have likely acquired these via horizontal gene transfer from eukaryotes. Finally, we propose a potential candidate for the elusive enzyme performing C-3 ketoreduction (ERG27 equivalent) in land plants and probably in other eukaryotic phyla. PMID:20333205

  15. Repetitive DNA in eukaryotic genomes.

    PubMed

    Biscotti, Maria Assunta; Olmo, Ettore; Heslop-Harrison, J S Pat

    2015-09-01

    Repetitive DNA--sequence motifs repeated hundreds or thousands of times in the genome--makes up the major proportion of all the nuclear DNA in most eukaryotic genomes. However, the significance of repetitive DNA in the genome is not completely understood, and it has been considered to have both structural and functional roles, or perhaps even no essential role. High-throughput DNA sequencing reveals huge numbers of repetitive sequences. Most bioinformatic studies focus on low-copy DNA including genes, and hence, the analyses collapse repeats in assemblies presenting only one or a few copies, often masking out and ignoring them in both DNA and RNA read data. Chromosomal studies are proving vital to examine the distribution and evolution of sequences because of the challenges of analysis of sequence data. Many questions are open about the origin, evolutionary mode and functions that repetitive sequences might have in the genome. Some, the satellite DNAs, are present in long arrays of similar motifs at a small number of sites, while others, particularly the transposable elements (DNA transposons and retrotranposons), are dispersed over regions of the genome; in both cases, sequence motifs may be located at relatively specific chromosome domains such as centromeres or subtelomeric regions. Here, we overview a range of works involving detailed characterization of the nature of all types of repetitive sequences, in particular their organization, abundance, chromosome localization, variation in sequence within and between chromosomes, and, importantly, the investigation of their transcription or expression activity. Comparison of the nature and locations of sequences between more, and less, related species is providing extensive information about their evolution and amplification. Some repetitive sequences are extremely well conserved between species, while others are among the most variable, defining differences between even closely relative species. These data suggest

  16. An experimentally tested scenario for the structural evolution of eukaryotic Cys2His2 zinc fingers from eubacterial ros homologs.

    PubMed

    Netti, Fortuna; Malgieri, Gaetano; Esposito, Sabrina; Palmieri, Maddalena; Baglivo, Ilaria; Isernia, Carla; Omichinski, James G; Pedone, Paolo V; Lartillot, Nicolas; Fattorusso, Roberto

    2013-07-01

    The exact evolutionary origin of the zinc finger (ZF) domain is unknown, as it is still not clear from which organisms it was first derived. However, the unique features of the ZF domains have made it very easy for evolution to tinker with them in a number of different manners, including their combination, variation of their number by unequal crossing-over or tandem duplication and tuning of their affinity for specific DNA sequence motifs through point substitutions. Classical Cys2His2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys2His2 ZF domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens and recently characterized. The present work focuses on the evolution of the classical ZF domains with the goal of trying to determine whether eukaryotic ZFs have evolved from the prokaryotic Ros-like proteins. Our results, based on computational and experimental data, indicate that a single insertion of three amino acids in the short loop that separates the β-sheet from the α-helix of the Ros protein is sufficient to induce a structural transition from a Ros like to an eukaryotic-ZF like structure. This observation provides evidence for a structurally plausible and parsimonious scenario of fold evolution, giving a structural basis to the hypothesis of a horizontal gene transfer (HGT) from bacteria to eukaryotes. PMID:23576569

  17. New cyclodextrin derivative containing poly(L-lysine) dendrons for gene and drug co-delivery.

    PubMed

    Ma, Dong; Zhang, Hong-Bin; Chen, Yu-Yun; Lin, Jian-Tao; Zhang, Li-Ming

    2013-09-01

    To develop a multifunctional polymeric carrier for gene and drug co-delivery, a new cyclodextrin derivative containing poly(L-lysine) dendrons was prepared by the click conjugation of per-6-azido-β-cyclodextrin with propargyl focal point poly(L-lysine) dendron of third generation and then characterized by FTIR, (1)H NMR, and GPC analyses. It was found that such a conjugate could form colloidally stable nanocomplexes with plasmid DNA in aqueous system and exhibited high gene transfection efficiency. Moreover, it could load efficiently methotrexate drug with anticancer activity and showed a sustained release behavior. Different from commonly used amphiphilic copolymers with cationic character, the as obtained cyclodextrin derivative may be used directly for the combinatorial delivery of nucleic acid and lipophilic anticancer drugs without a complicated micellization process. PMID:23769303

  18. The new phylogeny of eukaryotes.

    PubMed

    Philippe, H; Germot, A; Moreira, D

    2000-12-01

    Molecular phylogeny has been regarded as the ultimate tool for the reconstruction of relationships among eukaryotes-especially the different protist groups-given the difficulty in interpreting morphological data from an evolutionary point of view. In fact, the use of ribosomal RNA as a marker has provided the first well resolved eukaryotic phylogenies, leading to several important evolutionary hypotheses. The most significant is that several early-emerging, amitochondriate lineages, are living relics from the early times of eukaryotic evolution. The use of alternative protein markers and the recognition of several molecular phylogeny reconstruction artefacts, however, have strongly challenged these ideas. The putative early emerging lineages have been demonstrated as late-emerging ones, artefactually misplaced to the base of the tree. The present state of eukaryotic evolution is best described by a multifurcation, in agreement with the 'big bang' hypothesis that assumes a rapid diversification of the major eukaryotic phyla. For further resolution, the analysis of genomic data through improved phylogenetic methods will be required.

  19. Live-cell, temporal gene expression analysis of osteogenic differentiation in adipose-derived stem cells.

    PubMed

    Desai, Hetal V; Voruganti, Indu S; Jayasuriya, Chathuraka; Chen, Qian; Darling, Eric M

    2014-03-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3-5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs.

  20. Live-Cell, Temporal Gene Expression Analysis of Osteogenic Differentiation in Adipose-Derived Stem Cells

    PubMed Central

    Desai, Hetal V.; Voruganti, Indu S.; Jayasuriya, Chathuraka; Chen, Qian

    2014-01-01

    Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3–5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs. PMID:24367991

  1. Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

    PubMed Central

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-01-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

  2. Baculovirus: an insect-derived vector for diverse gene transfer applications.

    PubMed

    Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

    2013-04-01

    Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered.

  3. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-06-24

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies.

  4. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-01-01

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies. PMID:27572976

  5. The revised classification of eukaryotes.

    PubMed

    Adl, Sina M; Simpson, Alastair G B; Lane, Christopher E; Lukeš, Julius; Bass, David; Bowser, Samuel S; Brown, Matthew W; Burki, Fabien; Dunthorn, Micah; Hampl, Vladimir; Heiss, Aaron; Hoppenrath, Mona; Lara, Enrique; Le Gall, Line; Lynn, Denis H; McManus, Hilary; Mitchell, Edward A D; Mozley-Stanridge, Sharon E; Parfrey, Laura W; Pawlowski, Jan; Rueckert, Sonja; Shadwick, Laura; Shadwick, Lora; Schoch, Conrad L; Smirnov, Alexey; Spiegel, Frederick W

    2012-09-01

    This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information.

  6. Ethanol upregulates NMDA receptor subunit gene expression in human embryonic stem cell-derived cortical neurons.

    PubMed

    Xiang, Yangfei; Kim, Kun-Yong; Gelernter, Joel; Park, In-Hyun; Zhang, Huiping

    2015-01-01

    Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.

  7. Synaptic view of eukaryotic cell

    NASA Astrophysics Data System (ADS)

    Baluška, František; Mancuso, Stefano

    2014-10-01

    Synapses are stable adhesive domains between two neighbouring cells of the multicellular organisms which serve for cell-cell communication as well as for information processing and storing. The synaptic concept was developed over more than 100 years specifically for neuronal cell-cell communication. In the last ten years, this concept was adapted to embrace other cell-cell communication phenomena. Here, we focus on the recently emerged phagocytic synapse and propose new endosymbiotic synapses and "intracellular organellar synapses". All these synapses of eukaryotic cells are in a good position to explain the high capacity of eukaryotic cells for integration of diverse signalling inputs into coherent cellular behaviour.

  8. Self-splicing group I introns in eukaryotic viruses.

    PubMed

    Yamada, T; Tamura, K; Aimi, T; Songsri, P

    1994-07-11

    We report the occurrence of self-splicing group I introns in viruses that infect the eukaryotic green alga Chlorella. The introns contained all the conserved features of primary sequence and secondary structure previously described for the group IB introns. The Chlorella viral introns (approximately 400 nt) self-spliced in vitro, yielding the typical group I intron splicing intermediates and products. Contrasting to eukaryotic nuclear group I introns, all of which are located in the rRNA genes, these introns were inserted in genes encoding proteins. In one case, the exons encoded a protein showing significant homology to the eukaryotic transcription factor SII (TFIIS), which may be important for viral gene expression. In another case, the gene for the open reading frame (ORF) of a 14.2 kDa polypeptide with unknown functions contained the intron. Scattered distribution of these introns among the viral species and their structural similarity to the group I introns of algae and protists indicated horizontal intron transmission. These eukaryotic viral introns offer an opportunity to understand how group I introns reach organisms of different phylogenetic kingdoms.

  9. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    SciTech Connect

    Vadas, M.A.

    1982-02-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F/sub 1/ mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR..-->..F/sub 1/ were high responders and EO-LR..-->..F/sub 1/ were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy.

  10. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    PubMed Central

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  11. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    PubMed

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  12. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

    PubMed Central

    Kwon, Jin Seon; Park, Seung Hun; Baek, Ji Hye; Dung, Truong Minh; Kim, Sung Won; Min, Byoung Hyun; Kim, Jae Ho; Kim, Moon Suk

    2016-01-01

    Human turbinate mesenchymal stromal cells (hTMSCs) are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI) as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs) were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy. PMID:26783402

  13. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

    PubMed Central

    Forsberg, Kevin J.; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D.

    2015-01-01

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. PMID:26546427

  14. An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

    PubMed Central

    Jones, R M; Branda, J; Johnston, K A; Polymenis, M; Gadd, M; Rustgi, A; Callanan, L; Schmidt, E V

    1996-01-01

    The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation. PMID:8756633

  15. Silencing of host genes directed by virus-derived short interfering RNAs in Caenorhabditis elegans.

    PubMed

    Guo, Xunyang; Li, Wan-Xiang; Lu, Rui

    2012-11-01

    Small interfering RNAs (siRNAs) processed from viral replication intermediates by RNase III-like enzyme Dicer guide sequence-specific antiviral silencing in fungi, plants, and invertebrates. In plants, virus-derived siRNAs (viRNAs) can target and silence cellular transcripts and, in some cases, are responsible for the induction of plant diseases. Currently it remains unclear whether viRNAs are also capable of modulating the expression of cellular genes in the animal kingdom, although animal virus-encoded microRNAs (miRNAs) are known to guide efficient silencing of host genes, thereby facilitating virus replication. In this report, we showed that viRNAs derived from a modified nodavirus triggered potent silencing of homologous cellular transcripts produced by the endogenous gene or transgene in the nematode worm Caenorhabditis elegans. Like that found in plants, virus-induced gene silencing (VIGS) in C. elegans also involves RRF-1, a worm RNA-dependent RNA polymerase (RdRP) that is known to produce single-stranded secondary siRNAs in a Dicer-independent manner. We further demonstrated that VIGS in C. elegans is inheritable, suggesting that VIGS has the potential to generate profound epigenetic consequences in future generations. Altogether, these findings, for the first time, confirmed that viRNAs have the potential to modulate host gene expression in the animal kingdom. Most importantly, the success in uncoupling the trigger and the target of the antiviral silencing would allow for the exploration of novel features of virus-host interactions mediated by viRNAs in the animal kingdom. PMID:22896621

  16. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier

    PubMed Central

    Peng, Yu-Shiang; Lai, Po-Liang; Peng, Sydney; Wu, His-Chin; Yu, Siang; Tseng, Tsan-Yun; Wang, Li-Fang; Chu, I-Ming

    2014-01-01

    Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson’s disease. PMID:25061293

  17. Eukaryotic organisms in Proterozoic oceans.

    PubMed

    Knoll, A H; Javaux, E J; Hewitt, D; Cohen, P

    2006-06-29

    The geological record of protists begins well before the Ediacaran and Cambrian diversification of animals, but the antiquity of that history, its reliability as a chronicle of evolution and the causal inferences that can be drawn from it remain subjects of debate. Well-preserved protists are known from a relatively small number of Proterozoic formations, but taphonomic considerations suggest that they capture at least broad aspects of early eukaryotic evolution. A modest diversity of problematic, possibly stem group protists occurs in ca 1800-1300 Myr old rocks. 1300-720 Myr fossils document the divergence of major eukaryotic clades, but only with the Ediacaran-Cambrian radiation of animals did diversity increase within most clades with fossilizable members. While taxonomic placement of many Proterozoic eukaryotes may be arguable, the presence of characters used for that placement is not. Focus on character evolution permits inferences about the innovations in cell biology and development that underpin the taxonomic and morphological diversification of eukaryotic organisms.

  18. The early eukaryotic fossil record.

    PubMed

    Javaux, Emmanuelle J

    2007-01-01

    The Precambrian era records the evolution of the domain Eucarya. Although the taxonomy of fossils is often impossible to resolve beyond the level of domain, their morphology and chemistry indicate the evolution of major biological innovations. The late Archean record for eukaryotes is limited to trace amounts of biomarkers. Morphological evidence appears in late Paleoproterozoic and early Mesoproterozoic (1800-1300 Ma) rocks. The moderate diversity of preservable eukaryotic organisms includes cell walls without surface ornament (but with complex ultrastructure), with regularly distributed surface ornamentation, and with irregularly or regularly arranged processes. Collectively, these fossils suggest that eukaryotes with flexible membranes and cytoskeletons existed in mid-Proterozoic oceans. The late Mesoproterozoic-early Neoproterozoic (1300-750 Ma) is a time of diversification and evolution when direct evidence for important biological innovations occurs in the fossil record such as multicellularity, sex, photosynthesis, biomineralization, predation, and heterotrophy. Members of extant clades can be recognized and include bangiophyte red algae, xanthophyte algae, cladophorale green algae, euglyphid, lobose, and filose amoebae and possible fungi. In the late Neoproterozoic, besides more diversification of ornamented fossils, florideophyte red algae and brown algae diversify, and animals take the stage. The record of biological innovations documented by the fossils shows that eukaryotes had evolved most cytological and molecular complexities very early in the Proterozoic but environmental conditions delayed their diversification within clades until oxygen level and predation pressure increased significantly. PMID:17977455

  19. Eukaryotic organisms in Proterozoic oceans

    PubMed Central

    Knoll, A.H; Javaux, E.J; Hewitt, D; Cohen, P

    2006-01-01

    The geological record of protists begins well before the Ediacaran and Cambrian diversification of animals, but the antiquity of that history, its reliability as a chronicle of evolution and the causal inferences that can be drawn from it remain subjects of debate. Well-preserved protists are known from a relatively small number of Proterozoic formations, but taphonomic considerations suggest that they capture at least broad aspects of early eukaryotic evolution. A modest diversity of problematic, possibly stem group protists occurs in ca 1800–1300 Myr old rocks. 1300–720 Myr fossils document the divergence of major eukaryotic clades, but only with the Ediacaran–Cambrian radiation of animals did diversity increase within most clades with fossilizable members. While taxonomic placement of many Proterozoic eukaryotes may be arguable, the presence of characters used for that placement is not. Focus on character evolution permits inferences about the innovations in cell biology and development that underpin the taxonomic and morphological diversification of eukaryotic organisms. PMID:16754612

  20. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments. PMID:27026484

  1. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  2. Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons

    PubMed Central

    Heman-Ackah, Sabrina Mahalia; Bassett, Andrew Roger; Wood, Matthew John Andrew

    2016-01-01

    The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases. The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases. Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons. This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis. PMID:27341390

  3. Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility.

    PubMed

    Ju, Jia; Huan, Meng-Lei; Wan, Ning; Hou, Yi-Lin; Ma, Xi-Xi; Jia, Yi-Yang; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2016-05-15

    Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery. PMID:27072908

  4. Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers.

    PubMed

    Pandey, Prashant K; Siddharth, Jay; Verma, Pankaj; Bavdekar, Ashish; Patole, Milind S; Shouche, Yogesh S

    2012-06-01

    The micro-eukaryotic diversity from the human gut was investigated using universal primers directed towards 18S rRNA gene, fecal samples being the source of DNA. The subjects in this study included two breast-fed and two formula-milk-fed infants and their mothers. The study revealed that the infants did not seem to harbour any microeukaryotes in their gut. In contrast, there were distinct eukaryotic microbiota present in the mothers. The investigation is the first of its kind in the comparative study of the human feces to reveal the presence of micro-eukaryotic diversity variance in infants and adults from the Indian subcontinent. The micro-eukaryotes encountered during the investigation include known gut colonizers like Blastocystis and some fungi species. Some of these micro-eukaryotes have been speculated to be involved in clinical manifestations of various diseases. The study is an attempt to highlight the importance of micro-eukaryotes in the human gut.

  5. Signaling and Gene Regulatory Networks Governing Definitive Endoderm Derivation From Pluripotent Stem Cells.

    PubMed

    Mohammadnia, Abdulshakour; Yaqubi, Moein; Pourasgari, Farzaneh; Neely, Eric; Fallahi, Hossein; Massumi, Mohammad

    2016-09-01

    The generation of definitive endoderm (DE) from pluripotent stem cells (PSCs) is a fundamental stage in the formation of highly organized visceral organs, such as the liver and pancreas. Currently, there is a need for a comprehensive study that illustrates the involvement of different signaling pathways and their interactions in the derivation of DE cells from PSCs. This study aimed to identify signaling pathways that have the greatest influence on DE formation using analyses of transcriptional profiles, protein-protein interactions, protein-DNA interactions, and protein localization data. Using this approach, signaling networks involved in DE formation were constructed using systems biology and data mining tools, and the validity of the predicted networks was confirmed experimentally by measuring the mRNA levels of hub genes in several PSCs-derived DE cell lines. Based on our analyses, seven signaling pathways, including the BMP, ERK1-ERK2, FGF, TGF-beta, MAPK, Wnt, and PIP signaling pathways and their interactions, were found to play a role in the derivation of DE cells from PSCs. Lastly, the core gene regulatory network governing this differentiation process was constructed. The results of this study could improve our understanding surrounding the efficient generation of DE cells for the regeneration of visceral organs. J. Cell. Physiol. 231: 1994-2006, 2016. © 2016 Wiley Periodicals, Inc. PMID:26755186

  6. Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period.

    PubMed

    Nakamura, Keigo; Kusama, Kazuya; Bai, Rulan; Sakurai, Toshihiro; Isuzugawa, Kazuto; Godkin, James D; Suda, Yoshihito; Imakawa, Kazuhiko

    2016-01-01

    Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to

  7. Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period

    PubMed Central

    Nakamura, Keigo; Kusama, Kazuya; Bai, Rulan; Sakurai, Toshihiro; Isuzugawa, Kazuto; Godkin, James D.; Suda, Yoshihito; Imakawa, Kazuhiko

    2016-01-01

    Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to

  8. Silence of the strands: RNA interference in eukaryotic pathogens.

    PubMed

    Cottrell, Tricia R; Doering, Tamara L

    2003-01-01

    Double-stranded (ds) RNA interference (RNAi) is a recent technological advance that enables researchers to reduce gene expression at the post-transcriptional level. This form of RNA silencing is initiated by dsRNA, expressed in or introduced into a cell of interest, which triggers homology-dependent degradation of the corresponding mRNA. This versatile technique has remarkable promise as a tool for the study of eukaryotic pathogens. Protozoan parasites and pathogenic fungi often resist manipulation using standard molecular genetic approaches. Researchers studying these organisms need flexible molecular tools, particularly to exploit newly sequenced genomes; this review offers a practical guide to establishing RNAi in pathogenic eukaryotes.

  9. Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

    PubMed Central

    Andreeva, Alexandra V; Kutuzov, Mikhail A

    2004-01-01

    Background In eukaryotes, PPP (protein phosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases. Results We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I

  10. Gene Expression Analysis of HCT116 Colon Tumor-derived Cells Treated with the Polyamine Analog PG-11047

    PubMed Central

    IGNATENKO, NATALIA A.; YERUSHALMI, HAGIT F.; PANDEY, RITU; KACHEL, KAREN L.; STRINGER, DAVID E.; MARTON, LAURENCE J.; GERNER, EUGENE W.

    2013-01-01

    Background The conformationally restricted polyamine analog PG-11047 has significant growth inhibitory activity against prostate and lung cancer cell lines and is currently under evaluation in several clinical trials, both alone and in combination with other drugs, for the treatment of relapsed or refractory cancer. The objective of this study was to identify the molecular signature of genes responsive to PG-11047 treatment and the biochemical effects of this drug in the HCT116 colon cancer cell line. Materials and Methods Gene expression analysis was performed using Affymetrix GeneChip human genome U133 Plus 2.0 arrays. Changes in protein expression were evaluated using 2D polyacrylamide gels followed by LCMS/MS. Results Treatment of cells with PG-11047 at concentrations ranging from 0.1 to 10 μM caused inhibition of cell growth. The activity of PG-11047 was found to correlate with its transcriptional effects on cell cycle control, focal adhesion, adherent and gap junction genes, MAPK-, Wnt- and, TGF-β signaling pathways, transport and DNA/RNA transcription factor genes. PG-11047 caused depletion of polyamine pools. Proteomics analysis showed that PG-11047 restricts the modification of eukaryotic translation initiation factor 5A (eIF5A), resulting in suppression of general protein synthesis in PG-11047-treated cells. Conclusion These data show that PG-11047 has a broad spectrum of anticancer activity in colon cancer cells. PMID:19487545

  11. Differentiation of AFS cells derived from the EGFP gene transgenic porcine fetuses.

    PubMed

    Zheng, Yue-Mao; Dang, Yong-Hui; Xu, Yong-Ping; Sai, Wu-Jia-Fu; An, Zhi-Xing

    2011-08-01

    We have obtained the EGFP (enhanced green fluorescence protein) gene transgenic porcine fetuses before. The aims of this study were (i) to determine whether stem cells could be isolated from amniotic fluid of the transgenic porcine fetuses, and (ii) to determine if these stem cells could express EGFP and differentiate in vitro. The results demonstrated that stem cells could be isolated from amniotic fluid of the EGFP gene transgenic porcine fetuses and could express EGFP and differentiate in vitro. Undifferentiated AFSs (amniotic fluid-derived stem cells) expressed POU5F1, THY1 and SOX2, while the following differentiation cells expressed markers for chondrogenic (COL2A1), osteogenic (osteocalcin and osteonectin) and neurogenic cells such as astrocyte (GFAP), oligodendrocyte (GALC) and neuron (NF, ENO2 and MAP).

  12. Lymphoid Regeneration from Gene Corrected SCID-X1 Patient Derived iPSC

    PubMed Central

    Menon, T; Firth, AL; Scripture-Adams, D.D.; Galic, Z; Qualls, SJ; Gilmore, WB; Ke, E; Singer, O; Anderson, LS; Bornzin, AR; Alexander, IE; Zack, JA; Verma, IM

    2015-01-01

    Summary X-Linked severe combined immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted lifespan of less than one year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ) leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic patient-specific mutant and gene corrected iPSC lines. While the patient-derived mutant iPSC have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSC can additionally generate mature NK-cells and T-cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 patients. PMID:25772073

  13. Lymphoid regeneration from gene-corrected SCID-X1 subject-derived iPSCs.

    PubMed

    Menon, Tushar; Firth, Amy L; Scripture-Adams, Deirdre D; Galic, Zoran; Qualls, Susan J; Gilmore, William B; Ke, Eugene; Singer, Oded; Anderson, Leif S; Bornzin, Alexander R; Alexander, Ian E; Zack, Jerome A; Verma, Inder M

    2015-04-01

    X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects. PMID:25772073

  14. OrthoMCL: identification of ortholog groups for eukaryotic genomes.

    PubMed

    Li, Li; Stoeckert, Christian J; Roos, David S

    2003-09-01

    The identification of orthologous groups is useful for genome annotation, studies on gene/protein evolution, comparative genomics, and the identification of taxonomically restricted sequences. Methods successfully exploited for prokaryotic genome analysis have proved difficult to apply to eukaryotes, however, as larger genomes may contain multiple paralogous genes, and sequence information is often incomplete. OrthoMCL provides a scalable method for constructing orthologous groups across multiple eukaryotic taxa, using a Markov Cluster algorithm to group (putative) orthologs and paralogs. This method performs similarly to the INPARANOID algorithm when applied to two genomes, but can be extended to cluster orthologs from multiple species. OrthoMCL clusters are coherent with groups identified by EGO, but improved recognition of "recent" paralogs permits overlapping EGO groups representing the same gene to be merged. Comparison with previously assigned EC annotations suggests a high degree of reliability, implying utility for automated eukaryotic genome annotation. OrthoMCL has been applied to the proteome data set from seven publicly available genomes (human, fly, worm, yeast, Arabidopsis, the malaria parasite Plasmodium falciparum, and Escherichia coli). A Web interface allows queries based on individual genes or user-defined phylogenetic patterns (http://www.cbil.upenn.edu/gene-family). Analysis of clusters incorporating P. falciparum genes identifies numerous enzymes that were incompletely annotated in first-pass annotation of the parasite genome.

  15. RNA Export through the NPC in Eukaryotes

    PubMed Central

    Okamura, Masumi; Inose, Haruko; Masuda, Seiji

    2015-01-01

    In eukaryotic cells, RNAs are transcribed in the nucleus and exported to the cytoplasm through the nuclear pore complex. The RNA molecules that are exported from the nucleus into the cytoplasm include messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), micro RNAs (miRNAs), and viral mRNAs. Each RNA is transported by a specific nuclear export receptor. It is believed that most of the mRNAs are exported by Nxf1 (Mex67 in yeast), whereas rRNAs, snRNAs, and a certain subset of mRNAs are exported in a Crm1/Xpo1-dependent manner. tRNAs and miRNAs are exported by Xpot and Xpo5. However, multiple export receptors are involved in the export of some RNAs, such as 60S ribosomal subunit. In addition to these export receptors, some adapter proteins are required to export RNAs. The RNA export system of eukaryotic cells is also used by several types of RNA virus that depend on the machineries of the host cell in the nucleus for replication of their genome, therefore this review describes the RNA export system of two representative viruses. We also discuss the NPC anchoring-dependent mRNA export factors that directly recruit specific genes to the NPC. PMID:25802992

  16. Viruses and viruslike particles of eukaryotic algae.

    PubMed Central

    Van Etten, J L; Lane, L C; Meints, R H

    1991-01-01

    Until recently there was little interest or information on viruses and viruslike particles of eukaryotic algae. However, this situation is changing. In the past decade many large double-stranded DNA-containing viruses that infect two culturable, unicellular, eukaryotic green algae have been discovered. These viruses can be produced in large quantities, assayed by plaque formation, and analyzed by standard bacteriophage techniques. The viruses are structurally similar to animal iridoviruses, their genomes are similar to but larger (greater than 300 kbp) than that of poxviruses, and their infection process resembles that of bacteriophages. Some of the viruses have DNAs with low levels of methylated bases, whereas others have DNAs with high concentrations of 5-methylcytosine and N6-methyladenine. Virus-encoded DNA methyltransferases are associated with the methylation and are accompanied by virus-encoded DNA site-specific (restriction) endonucleases. Some of these enzymes have sequence specificities identical to those of known bacterial enzymes, and others have previously unrecognized specificities. A separate rod-shaped RNA-containing algal virus has structural and nucleotide sequence affinities to higher plant viruses. Quite recently, viruses have been associated with rapid changes in marine algal populations. In the next decade we envision the discovery of new algal viruses, clarification of their role in various ecosystems, discovery of commercially useful genes in these viruses, and exploitation of algal virus genetic elements in plant and algal biotechnology. Images PMID:1779928

  17. The expanding roles of the ghrelin-gene derived peptide obestatin in health and disease.

    PubMed

    Seim, Inge; Walpole, Carina; Amorim, Laura; Josh, Peter; Herington, Adrian; Chopin, Lisa

    2011-06-20

    Obestatin is a 23 amino acid, ghrelin gene-derived peptide hormone produced in the stomach and a range of other tissues throughout the body. While it was initially reported that obestatin opposed the actions of ghrelin with regards to appetite and food intake, it is now clear that obestatin is not an endogenous ghrelin antagonist, but it is a multi-functional peptide hormone in its own right. In this review we will discuss the controversies associated with the discovery of obestatin and explore emerging central and peripheral roles of obestatin, which includes adipogenesis, pancreatic homeostasis and cancer.

  18. HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types

    PubMed Central

    Van Damme, Ellen; Thys, Kim; Tuefferd, Marianne; Van Hove, Carl; Aerssens, Jeroen; Van Loock, Marnix

    2016-01-01

    Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential

  19. Derived variants at six genes explain nearly half of size reduction in dog breeds.

    PubMed

    Rimbault, Maud; Beale, Holly C; Schoenebeck, Jeffrey J; Hoopes, Barbara C; Allen, Jeremy J; Kilroy-Glynn, Paul; Wayne, Robert K; Sutter, Nathan B; Ostrander, Elaine A

    2013-12-01

    Selective breeding of dogs by humans has generated extraordinary diversity in body size. A number of multibreed analyses have been undertaken to identify the genetic basis of this diversity. We analyzed four loci discovered in a previous genome-wide association study that used 60,968 SNPs to identify size-associated genomic intervals, which were too large to assign causative roles to genes. First, we performed fine-mapping to define critical intervals that included the candidate genes GHR, HMGA2, SMAD2, and STC2, identifying five highly associated markers at the four loci. We hypothesize that three of the variants are likely to be causative. We then genotyped each marker, together with previously reported size-associated variants in the IGF1 and IGF1R genes, on a panel of 500 domestic dogs from 93 breeds, and identified the ancestral allele by genotyping the same markers on 30 wild canids. We observed that the derived alleles at all markers correlated with reduced body size, and smaller dogs are more likely to carry derived alleles at multiple markers. However, breeds are not generally fixed at all markers; multiple combinations of genotypes are found within most breeds. Finally, we show that 46%-52.5% of the variance in body size of dog breeds can be explained by seven markers in proximity to exceptional candidate genes. Among breeds with standard weights <41 kg (90 lb), the genotypes accounted for 64.3% of variance in weight. This work advances our understanding of mammalian growth by describing genetic contributions to canine size determination in non-giant dog breeds.

  20. Derived variants at six genes explain nearly half of size reduction in dog breeds

    PubMed Central

    Rimbault, Maud; Beale, Holly C.; Schoenebeck, Jeffrey J.; Hoopes, Barbara C.; Allen, Jeremy J.; Kilroy-Glynn, Paul; Wayne, Robert K.; Sutter, Nathan B.; Ostrander, Elaine A.

    2013-01-01

    Selective breeding of dogs by humans has generated extraordinary diversity in body size. A number of multibreed analyses have been undertaken to identify the genetic basis of this diversity. We analyzed four loci discovered in a previous genome-wide association study that used 60,968 SNPs to identify size-associated genomic intervals, which were too large to assign causative roles to genes. First, we performed fine-mapping to define critical intervals that included the candidate genes GHR, HMGA2, SMAD2, and STC2, identifying five highly associated markers at the four loci. We hypothesize that three of the variants are likely to be causative. We then genotyped each marker, together with previously reported size-associated variants in the IGF1 and IGF1R genes, on a panel of 500 domestic dogs from 93 breeds, and identified the ancestral allele by genotyping the same markers on 30 wild canids. We observed that the derived alleles at all markers correlated with reduced body size, and smaller dogs are more likely to carry derived alleles at multiple markers. However, breeds are not generally fixed at all markers; multiple combinations of genotypes are found within most breeds. Finally, we show that 46%–52.5% of the variance in body size of dog breeds can be explained by seven markers in proximity to exceptional candidate genes. Among breeds with standard weights <41 kg (90 lb), the genotypes accounted for 64.3% of variance in weight. This work advances our understanding of mammalian growth by describing genetic contributions to canine size determination in non-giant dog breeds. PMID:24026177

  1. Compositional differences within and between eukaryotic genomes.

    PubMed

    Karlin, S; Mrázek, J

    1997-09-16

    Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is rho*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size >/=50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection (rho*XY) as the genome signature. This paper identifies rho*XY extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.

  2. Evolution: Steps on the road to eukaryotes

    NASA Astrophysics Data System (ADS)

    Embley, T. Martin; Williams, Tom A.

    2015-05-01

    A new archaeal phylum represents the closest known relatives of eukaryotes, the group encompassing all organisms that have nucleated cells. The discovery holds promise for a better understanding of eukaryotic origins. See Article p.173

  3. Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae).

    PubMed Central

    Cavalier-Smith, T

    2003-01-01

    Chloroplasts originated just once, from cyanobacteria enslaved by a biciliate protozoan to form the plant kingdom (green plants, red and glaucophyte algae), but subsequently, were laterally transferred to other lineages to form eukaryote-eukaryote chimaeras or meta-algae. This process of secondary symbiogenesis (permanent merger of two phylogenetically distinct eukaryote cells) has left remarkable traces of its evolutionary role in the more complex topology of the membranes surrounding all non-plant (meta-algal) chloroplasts. It took place twice, soon after green and red algae diverged over 550 Myr ago to form two independent major branches of the eukaryotic tree (chromalveolates and cabozoa), comprising both meta-algae and numerous secondarily non-photosynthetic lineages. In both cases, enslavement probably began by evolving a novel targeting of endomembrane vesicles to the perialgal vacuole to implant host porter proteins for extracting photosynthate. Chromalveolates arose by such enslavement of a unicellular red alga and evolution of chlorophyll c to form the kingdom Chromista and protozoan infrakingdom Alveolata, which diverged from the ancestral chromalveolate chimaera. Cabozoa arose when the common ancestor of euglenoids and cercozoan chlorarachnean algae enslaved a tetraphyte green alga with chlorophyll a and b. I suggest that in cabozoa the endomembrane vesicles originally budded from the Golgi, whereas in chromalveolates they budded from the endoplasmic reticulum (ER) independently of Golgi-targeted vesicles, presenting a potentially novel target for drugs against alveolate Sporozoa such as malaria parasites and Toxoplasma. These hypothetical ER-derived vesicles mediated fusion of the perialgal vacuole and rough ER (RER) in the ancestral chromist, placing the former red alga within the RER lumen. Subsequently, this chimaera diverged to form cryptomonads, which retained the red algal nucleus as a nucleomorph (NM) with approximately 464 protein-coding genes

  4. Censusing marine eukaryotic diversity in the twenty-first century.

    PubMed

    Leray, Matthieu; Knowlton, Nancy

    2016-09-01

    The ocean constitutes one of the vastest and richest biomes on our planet. Most recent estimations, all based on indirect approaches, suggest that there are millions of marine eukaryotic species. Moreover, a large majority of these are small (less than 1 mm), cryptic and still unknown to science. However, this knowledge gap, caused by the lack of diagnostic morphological features in small organisms and the limited sampling of the global ocean, is currently being filled, thanks to new DNA-based approaches. The molecular technique of PCR amplification of homologous gene regions combined with high-throughput sequencing, routinely used to census unculturable prokaryotes, is now also being used to characterize whole communities of marine eukaryotes. Here, we review how this methodological advancement has helped to better quantify the magnitude and patterns of marine eukaryotic diversity, with an emphasis on taxonomic groups previously largely overlooked. We then discuss obstacles remaining to achieve a global understanding of marine eukaryotic diversity. In particular, we argue that 18S variable regions do not provide sufficient taxonomic resolution to census marine life, and suggest combining broad eukaryotic surveys targeting the 18S rRNA region with more taxon-focused analyses of hypervariable regions to improve our understanding of the diversity of species, the functional units of marine ecosystems.This article is part of the themed issue 'From DNA barcodes to biomes'. PMID:27481783

  5. Censusing marine eukaryotic diversity in the twenty-first century.

    PubMed

    Leray, Matthieu; Knowlton, Nancy

    2016-09-01

    The ocean constitutes one of the vastest and richest biomes on our planet. Most recent estimations, all based on indirect approaches, suggest that there are millions of marine eukaryotic species. Moreover, a large majority of these are small (less than 1 mm), cryptic and still unknown to science. However, this knowledge gap, caused by the lack of diagnostic morphological features in small organisms and the limited sampling of the global ocean, is currently being filled, thanks to new DNA-based approaches. The molecular technique of PCR amplification of homologous gene regions combined with high-throughput sequencing, routinely used to census unculturable prokaryotes, is now also being used to characterize whole communities of marine eukaryotes. Here, we review how this methodological advancement has helped to better quantify the magnitude and patterns of marine eukaryotic diversity, with an emphasis on taxonomic groups previously largely overlooked. We then discuss obstacles remaining to achieve a global understanding of marine eukaryotic diversity. In particular, we argue that 18S variable regions do not provide sufficient taxonomic resolution to census marine life, and suggest combining broad eukaryotic surveys targeting the 18S rRNA region with more taxon-focused analyses of hypervariable regions to improve our understanding of the diversity of species, the functional units of marine ecosystems.This article is part of the themed issue 'From DNA barcodes to biomes'.

  6. A search for extraterrestrial eukaryotes: physical and paleontological aspects.

    PubMed

    Chela-Flores, J

    1998-10-01

    Physical and biochemical aspects of a proposed search for extraterrestrial eukaryotes (SETE) are considered. Such a program should approach the distinction between a primitive eukaryote and an archaebacteria. The emphasis on gene silencing suggests a possible assay suitable for a robotic investigation of eukaryoticity, so as to be able to decide whether the first steps towards eukaryogenesis have been taken in an extraterrestrial planet, or satellite. The experiment would consist of searching for cellular division and the systematic related delay in replication of heterochromatic chromosome segments. It should be noticed that the direct search for a membrane-bounded set of chromosomes does not necessarily determine eukaryotic identity, as there are prokaryotes that have membrane-bounded nucleoids. A closer look at the protein fraction of chromatin (mainly histones) does not help either, as there are some eukaryotes that may lack histones; there are also some bacteria as well as archaebacteria with histone-like proteins in their nucleoids. Comments on the recent suggestion of possible environments for a SETE program are discussed: the deep crust of Mars, and the Jovian satellite Europa, provided the existence of an ocean under its ice-covered surface is confirmed by the current Galileo mission. PMID:9742730

  7. A search for extraterrestrial eukaryotes: physical and paleontological aspects.

    PubMed

    Chela-Flores, J

    1998-10-01

    Physical and biochemical aspects of a proposed search for extraterrestrial eukaryotes (SETE) are considered. Such a program should approach the distinction between a primitive eukaryote and an archaebacteria. The emphasis on gene silencing suggests a possible assay suitable for a robotic investigation of eukaryoticity, so as to be able to decide whether the first steps towards eukaryogenesis have been taken in an extraterrestrial planet, or satellite. The experiment would consist of searching for cellular division and the systematic related delay in replication of heterochromatic chromosome segments. It should be noticed that the direct search for a membrane-bounded set of chromosomes does not necessarily determine eukaryotic identity, as there are prokaryotes that have membrane-bounded nucleoids. A closer look at the protein fraction of chromatin (mainly histones) does not help either, as there are some eukaryotes that may lack histones; there are also some bacteria as well as archaebacteria with histone-like proteins in their nucleoids. Comments on the recent suggestion of possible environments for a SETE program are discussed: the deep crust of Mars, and the Jovian satellite Europa, provided the existence of an ocean under its ice-covered surface is confirmed by the current Galileo mission.

  8. Censusing marine eukaryotic diversity in the twenty-first century

    PubMed Central

    Knowlton, Nancy

    2016-01-01

    The ocean constitutes one of the vastest and richest biomes on our planet. Most recent estimations, all based on indirect approaches, suggest that there are millions of marine eukaryotic species. Moreover, a large majority of these are small (less than 1 mm), cryptic and still unknown to science. However, this knowledge gap, caused by the lack of diagnostic morphological features in small organisms and the limited sampling of the global ocean, is currently being filled, thanks to new DNA-based approaches. The molecular technique of PCR amplification of homologous gene regions combined with high-throughput sequencing, routinely used to census unculturable prokaryotes, is now also being used to characterize whole communities of marine eukaryotes. Here, we review how this methodological advancement has helped to better quantify the magnitude and patterns of marine eukaryotic diversity, with an emphasis on taxonomic groups previously largely overlooked. We then discuss obstacles remaining to achieve a global understanding of marine eukaryotic diversity. In particular, we argue that 18S variable regions do not provide sufficient taxonomic resolution to census marine life, and suggest combining broad eukaryotic surveys targeting the 18S rRNA region with more taxon-focused analyses of hypervariable regions to improve our understanding of the diversity of species, the functional units of marine ecosystems. This article is part of the themed issue ‘From DNA barcodes to biomes’. PMID:27481783

  9. Kinome profiling using peptide arrays in eukaryotic cells.

    PubMed

    Parikh, Kaushal; Peppelenbosch, Maikel P; Ritsema, Tita

    2009-01-01

    Over the last 10 years array and mass spectrometry technologies have enabled the determination of the transcriptome and proteome of biological and in particular eukaryotic systems. This information will likely be of significant value to our elucidation of the molecular mechanisms that govern eukaryotic physiology. However, an equally, if not more important goal, is to define those proteins that participate in signalling pathways that ultimately control cell fate. Enzymes that phosphorylate tyrosine, serine, and threonine residues on other proteins play a major role in signalling cascades that determine cell-cycle entry, and survival and differentiation fate in the tissues across the eukaryotic kingdoms. Knowing which signalling pathways are being used in these cells is of critical importance. Traditional genetic and biochemical approaches can certainly provide answers here, but for technical and practical reasons there is typically pursued one gene or pathway at a time. Thus, a more comprehensive approach is needed in order to reveal signalling pathways active in nucleated cells. Towards this end, kinome analysis techniques using peptide arrays have begun to be applied with substantial success in a variety of organisms from all major branches of eukaryotic life, generating descriptions of cellular signalling without a priori assumptions as to possibly effected pathways. The general procedure and analysis methods are very similar disregarding whether the primary source of the material is animal, plant, or fungal of nature and will be described in this chapter. These studies will help us better understand what signalling pathways are critical to controlling eukaryotic cell function.

  10. Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical review.

    PubMed

    Gutiérrez, Juan C; Amaro, Francisco; Martín-González, Ana

    2015-01-01

    This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported.

  11. Heavy metal whole-cell biosensors using eukaryotic microorganisms: an updated critical review

    PubMed Central

    Gutiérrez, Juan C.; Amaro, Francisco; Martín-González, Ana

    2015-01-01

    This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported. PMID:25750637

  12. New symbiotic hypothesis on the origin of eukaryotic flagella

    NASA Astrophysics Data System (ADS)

    Li, Jing Yan; Wu, Chuan Fen

    2005-07-01

    The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis’ 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes’ making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such

  13. New symbiotic hypothesis on the origin of eukaryotic flagella.

    PubMed

    Li, Jing Yan; Wu, Chuan Fen

    2005-07-01

    The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis' 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes' making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such

  14. Hydrogenosomes: eukaryotic adaptations to anaerobic environments.

    PubMed

    Hackstein, J H; Akhmanova, A; Boxma, B; Harhangi, H R; Voncken, F G

    1999-11-01

    Like mitochondria, hydrogenosomes compartmentalize crucial steps of eukaryotic energy metabolism; however, this compartmentalization differs substantially between mitochondriate aerobes and hydrogenosome-containing anaerobes. Because hydrogenosomes have arisen independently in different lineages of eukaryotic microorganisms, comparative analysis of the various types of hydrogenosomes can provide insights into the functional and evolutionary aspects of compartmentalized energy metabolism in unicellular eukaryotes.

  15. Defining DNA-based operational taxonomic units for microbial-eukaryote ecology.

    PubMed

    Caron, David A; Countway, Peter D; Savai, Pratik; Gast, Rebecca J; Schnetzer, Astrid; Moorthi, Stefanie D; Dennett, Mark R; Moran, Dawn M; Jones, Adriane C

    2009-09-01

    DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

  16. The revised classification of eukaryotes

    PubMed Central

    Adl, Sina M.; Simpson, Alastair. G.; Lane, Christopher E.; Lukeš, Julius; Bass, David; Bowser, Samuel S.; Brown, Matt; Burki, Fabien; Dunthorn, Micah; Hampl, Vladimir; Heiss, Aaron; Hoppenrath, Mona; Lara, Enrique; leGall, Line; Lynn, Denis H.; McManus, Hilary; Mitchell, Edward A. D.; Mozley-Stanridge, Sharon E.; Parfrey, Laura Wegener; Pawlowski, Jan; Rueckert, Sonja; Shadwick, Lora; Schoch, Conrad; Smirnov, Alexey; Spiegel, Frederick W.

    2012-01-01

    This revision of the classification of eukaryotes, which updates that of Adl et al. (2005), retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re-introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under-sampled areas and from environmental genomic information. PMID:23020233

  17. An investigation into eukaryotic pseudouridine synthases.

    PubMed

    King, Ross D; Lu, Chuan

    2014-08-01

    A common post-transcriptional modification of RNA is the conversion of uridine to its isomer pseudouridine. We investigated the biological significance of eukaryotic pseudouridine synthases using the yeast Saccharomyces cerevisiae. We conducted a comprehensive statistical analysis on growth data from automated perturbation (gene deletion) experiments, and used bi-logistic curve analysis to characterise the yeast phenotypes. The deletant strains displayed different alteration in growth properties, including in some cases enhanced growth and/or biphasic growth curves not seen in wild-type strains under matched conditions. These results demonstrate that disrupting pseudouridine synthases can have a significant qualitative effect on growth. We further investigated the significance of post-transcriptional pseudouridine modification through investigation of the scientific literature. We found that (1) In Toxoplasma gondii, a pseudouridine synthase gene is critical in cellular differentiation between the two asexual forms: Tachyzoites and bradyzoites; (2) Mutation of pseudouridine synthase genes has also been implicated in human diseases (mitochondrial myopathy and sideroblastic anemia (MLASA); dyskeratosis congenita). Taken together, these results are consistent with pseudouridine synthases having a Gene Ontology function of "biological regulation".

  18. Molecular marker-assisted alien gene introgression of Sr39 for wheat stem rust resistance derived from Aegilops speltoides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In wheat (Triticum aestivum L.), stem rust resistance gene Sr39, derived from Aegilops speltoides, is highly effective against multiple stem rust races including Ug99. However, the gene has not been used in wheat breeding because it is located on a large 2S chromosomal segment in the current transl...

  19. Replicating Damaged DNA in Eukaryotes

    PubMed Central

    Chatterjee, Nimrat; Siede, Wolfram

    2013-01-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail. PMID:24296172

  20. Replicating damaged DNA in eukaryotes.

    PubMed

    Chatterjee, Nimrat; Siede, Wolfram

    2013-12-01

    DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.

  1. Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells

    PubMed Central

    Meier, Elizabeth M.; Wu, Bin; Siddiqui, Aamir; Tepper, Donna G.; Longaker, Michael T.

    2016-01-01

    Background: Efforts have been made to engineer knee meniscus tissue for injury repair, yet most attempts have been unsuccessful. Creating a cell source that resembles the complex, heterogeneous phenotype of the meniscus cell remains difficult. Stem cell differentiation has been investigated, mainly using bone marrow mesenchymal cells and biochemical means for differentiation, resulting in no solution. Mechanical stimulation has been investigated to an extent with no conclusion. Here, we explore the potential for and effectiveness of mechanical stimulation to induce the meniscal phenotype in adipose-derived stromal cells. Methods: Human adipose-derived stromal cells were chosen for their fibrogenic nature and conduciveness for chondrogenesis. Biochemical and mechanical stimulation were investigated. Biochemical stimulation included fibrogenic and chondrogenic media. For mechanical stimulation, a custom-built device was used to apply constant, cyclical, uniaxial strain for up to 6 hours. Strain and frequency varied. Results: Under biochemical stimulation, both fibrogenic (collagen I, versican) and chondrogenic (collagen II, Sox9, aggrecan) genes were expressed by cells exposed to either fibrogenic or chondrogenic biochemical factors. Mechanical strain was found to preferentially promote fibrogenesis over chondrogenesis, confirming that tensile strain is an effective fibrogenic cue. Three hours at 10% strain and 1 Hz in chondrogenic media resulted in the highest expression of fibrochondrogenic genes. Although mechanical stimulation did not seem to affect protein level expression, biochemical means did affect protein level presence of collagen fibers. Conclusion: Mechanical stimulation can be a useful differentiation tool for mechanoresponsive cell types as long as biochemical factors are also integrated. PMID:27757329

  2. Assessment of Difference in Gene Expression Profile Between Embryos of Different Derivations

    PubMed Central

    Kwon, Sujin; Jeong, Sangkyun; Jeong, Young Sun; Park, Jung Sun; Cui, Xiang-Shun; Kim, Nam-Hyung

    2015-01-01

    Abstract Researchers have exerted sustained efforts to improve the viability of somatic cell nuclear transfer (SCNT) embryos, testing their experimental designs and probing the resultant embryos. However, the lack of a reliable method to estimate the efficacy of these experimental attempts is a chief hindrance to tackling the low-viability problem in SCNT. Here, we introduce a procedure that assesses the degree of difference in gene expression profiles (GEPs) of blastocysts from each other as a representative control of good quality. We first adapted a multiplex reverse transcription-polymerase chain reaction strategy to obtain GEPs for 15 reprogramming-related genes from single mouse blastocysts. GEPs of individual blastocysts displayed a broad range of variations, the extent of which was calculated using a weighted root mean square deviation (wRMSD). wRMSD-based quantitation of GEP difference (qGEP) found that GEP difference between in vivo–derived blastocysts (in vivo) and SCNT blastocysts was greater than the difference between in vivo blastocysts and in vitro–produced (IVP) blastocysts, demonstrating that the SCNT group was more distantly related to the in vivo group than the IVP group. Our qGEP approach for grading individual blastocysts would be useful for selecting a better protocol to derive embryos of better quality prior to field applications. PMID:25549061

  3. Increased CD44 gene expression in lymphocytes derived from Alzheimer disease patients.

    PubMed

    Uberti, D; Cenini, G; Bonini, S A; Barcikowska, M; Styczynska, M; Szybinska, A; Memo, M

    2010-01-01

    In this study, we demonstrated for the first time an increased CD44 gene expression in lymphocytes derived from Alzheimer's disease (AD) patients in comparison with healthy subjects. CD44 is a surface antigen expressed by cells of the immune and central nervous system as well as in a variety of other tissues. Functioning as adhesion molecule, CD44 is furthermore involved in driving immune response into infected tissues, including the CNS. We also found that lymphocytes of the same patients expressed significant levels of unfolded p53 isoform, confirming what we already demonstrated in fibroblasts and lymphocytes derived from other cohorts of AD patients. A correlation between p53 and CD44 expression has been well demonstrated in cancer cells, suggesting that CD44 could be a target gene of mutant p53, or either mutant p53 could lack its ability to negatively regulate CD44 expression. The contemporaneous increased expression of unfolded p53 and CD44 in AD lymphocytes may suggest that these two molecules cross-talk together participating in peripheral immune response during the development of the disease.

  4. MlNCD1: A novel Aegilops tauschii derived powdery mildew resistance gene identified in common wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Powdery mildew is a major fungal disease in wheat, especially in cool maritime climates. A novel Aegilops tauschii derived wheat powdery mildew resistance gene present in the germplasm line NC96BGTD1 was genetically characterized as a monogenic trait in field trials using F2 and F4-derived lines fr...

  5. A bacterial proteorhodopsin proton pump in marine eukaryotes.

    PubMed

    Slamovits, Claudio H; Okamoto, Noriko; Burri, Lena; James, Erick R; Keeling, Patrick J

    2011-01-01

    Proteorhodopsins are light-driven proton pumps involved in widespread phototrophy. Discovered in marine proteobacteria just 10 years ago, proteorhodopsins are now known to have been spread by lateral gene transfer across diverse prokaryotes, but are curiously absent from eukaryotes. In this study, we show that proteorhodopsins have been acquired by horizontal gene transfer from bacteria at least twice independently in dinoflagellate protists. We find that in the marine predator Oxyrrhis marina, proteorhodopsin is indeed the most abundantly expressed nuclear gene and its product localizes to discrete cytoplasmic structures suggestive of the endomembrane system. To date, photosystems I and II have been the only known mechanism for transducing solar energy in eukaryotes; however, it now appears that some abundant zooplankton use this alternative pathway to harness light to power biological functions.

  6. Defensins: antifungal lessons from eukaryotes

    PubMed Central

    Silva, Patrícia M.; Gonçalves, Sónia; Santos, Nuno C.

    2014-01-01

    Over the last years, antimicrobial peptides (AMPs) have been the focus of intense research toward the finding of a viable alternative to current antifungal drugs. Defensins are one of the major families of AMPs and the most represented among all eukaryotic groups, providing an important first line of host defense against pathogenic microorganisms. Several of these cysteine-stabilized peptides present a relevant effect against fungi. Defensins are the AMPs with the broader distribution across all eukaryotic kingdoms, namely, Fungi, Plantae, and Animalia, and were recently shown to have an ancestor in a bacterial organism. As a part of the host defense, defensins act as an important vehicle of information between innate and adaptive immune system and have a role in immunomodulation. This multidimensionality represents a powerful host shield, hard for microorganisms to overcome using single approach resistance strategies. Pathogenic fungi resistance to conventional antimycotic drugs is becoming a major problem. Defensins, as other AMPs, have shown to be an effective alternative to the current antimycotic therapies, demonstrating potential as novel therapeutic agents or drug leads. In this review, we summarize the current knowledge on some eukaryotic defensins with antifungal action. An overview of the main targets in the fungal cell and the mechanism of action of these AMPs (namely, the selectivity for some fungal membrane components) are presented. Additionally, recent works on antifungal defensins structure, activity, and cytotoxicity are also reviewed. PMID:24688483

  7. (Viruses of eukaryotic green algae): Performance report

    SciTech Connect

    Not Available

    1987-01-01

    The primary objective of this research was to develop the Chlorella-PBCV-1 virus system so that it can be used as a model system for studying gene expression in a photosynthetic eukaryote. Discoveries include the finding that morphologically similar, plaque forming, dsDNA containing viruses are common in nature and can be isolated readily from fresh water; the finding that all of these Chlorella viruses contain methylated bases which range in concentration from 0.1% to 47.5% mVdC and 0 to 37% mWdA and the discovery that infection with at least some of these viruses induces the appearance of DNA modification/restriction systems. 18 refs.

  8. [Structure and function of the eukaryotic ribosome].

    PubMed

    Bakowska-Zywicka, Kamilla; Twardowski, Tomasz

    2008-01-01

    The protein biosynthesis is a complicated process and not fully understood yet. According to smaller size and less complicated structure, understanding of prokaryotic 70S ribosomes is much more advanced. Eucaryotic 80S ribosomes are more complex and generate more difficulties in research. The morphology of 80S ribosome has been pretty well resolved and we know a lot about mechanism of functioning. Determination of the interactions between the ribosomes and the factors taking part in protein biosynthesis is still a great challenge. Dynamic changes of these interactions during particular steps of elongation cycle are quite difficult to understand. Conformational changes of the ribosome are of great functional and regulatory importance during protein biosynthesis. They are essential for the whole gene expression process. Only further research of the structure and function of the ribosome will lead us to knowledge about specificity of the mechanism of their action. In this article we present current opinions concerning structure and function of the eukaryotic ribosomes.

  9. Haloviruses of archaea, bacteria, and eukaryotes.

    PubMed

    Atanasova, Nina S; Oksanen, Hanna M; Bamford, Dennis H

    2015-06-01

    Hypersaline environments up to near saturation are rich reservoirs of extremophilic viruses. One milliliter of salt water may contain up to 10(9) viruses which can also be trapped inside salt crystals. To date, most of the ∼100 known halovirus isolates infect extremely halophilic archaea, although a few bacterial and eukaryotic viruses have also been described. These isolates comprise tailed and tailless icosahedral, pleomorphic, and lemon-shaped viruses which have been classified according to features such as host range, genome type, and replication. Recent studies have revealed that viruses can be grouped into a few structure-based viral lineages derived from a common ancestor based on conserved virion architectural principles and the major capsid protein fold.

  10. Cloning and expression in streptomyces lividans of antibiotic resistance genes derived from Escherichia coli.

    PubMed Central

    Schottel, J L; Bibb, M J; Cohen, S N

    1981-01-01

    Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment. Images PMID:7012122

  11. In silico prediction of tumor antigens derived from functional missense mutations of the cancer gene census

    PubMed Central

    Khalili, Jahan S.; Hanson, Russell W.; Szallasi, Zoltan

    2012-01-01

    Antigen-specific immune responses against peptides derived from missense gene mutations have been identified in multiple cancers. The application of personalized peptide vaccines based on the tumor mutation repertoire of each cancer patient is a near-term clinical reality. These peptides can be identified for pre-validation by leveraging the results of massive gene sequencing efforts in cancer. In this study, we utilized NetMHC 3.2 to predict nanomolar peptide binding affinity to 57 human HLA-A and B alleles. All peptides were derived from 5,685 missense mutations in 312 genes annotated as functionally relevant in the Cancer Genome Project. Of the 26,672,189 potential 8–11 mer peptide-HLA pairs evaluated, 0.4% (127,800) display binding affinities < 50 nM, predicting high affinity interactions. These peptides can be segregated into two groups based on the binding affinity to HLA proteins relative to germline-encoded sequences: peptides for which both the mutant and wild-type forms are high affinity binders, and peptides for which only the mutant form is a high affinity binder. Current evidence directs the attention to mutations that increase HLA binding affinity, as compared with cognate wild-type peptide sequences, as these potentially are more relevant for vaccine development from a clinical perspective. Our analysis generated a database including all predicted HLA binding peptides and the corresponding change in binding affinity as a result of point mutations. Our study constitutes a broad foundation for the development of personalized peptide vaccines that hone-in on functionally relevant targets in multiple cancers in individuals with diverse HLA haplotypes. PMID:23243591

  12. Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

    PubMed

    Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

    2015-06-01

    To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. PMID:25843855

  13. [The gene ontology and electro localization of ovine skin derived EST-SSR markers].

    PubMed

    Wang, Zun-Bao; Zhao, Zong-Sheng; Yu, Peng; Wu, Hong-Bin; Ban, Qian; Liang, Yao-Wei; Zheng, Wei

    2011-07-01

    Abstract: In order to study the potential gene function of ovine EST-SSR markers, nine original EST of Ovine Skin Derived polymorphic EST-SSR loci, which were developed in an early study by our lab, were ontology annotated and Electro localized. The results revealed that the original ESTs of the six loci had high homology with known genes and three of them probably played an important role in wool traits. Compared with its cDNA library, 8 loci were located on chromosomes of cattle. The homology of chromosomes between cattle and sheep was estimated based on the similarity coefficients calculated by positioning markers. Additionally, NJ clustering tree was establishedto serve for electro localization of ovine EST-SSR markers. Finally, 8 EST-SSR markers were successfully positioned on ovine chromosomes. The results from this study not only provide references for further studies on genetic mapping, in silico cloning of key genes for wool traits, but also are helpful to the researchs of chromosome evolution in animal.

  14. Variation characteristics of the nitrate reductase gene of key inbred maize lines and derived lines in China.

    PubMed

    Zhang, J J; Zhang, X Q; Liu, Y H; Liu, H M; Wang, Y B; Tian, M L; Huang, Y B

    2010-09-14

    Key inbred lines have played a fundamental role in maize genetics and breeding. Research on variation characteristics of key genes from key inbred lines and from derived lines is important for early identification and evaluation of inbred maize lines. The nitrate reductase (NR) gene, which plays a central role in nitrate acquisition, was the target gene for this research. Forty-one inbred maize lines were investigated, including four key inbred lines: Huangzaosi, Mo17, Dan340, and Ye478. Through multiple sequence alignment with the NR gene from B73, used as a control, we found that: 1) the NR gene of most inbred lines from Huangzaosi and from derived lines had two insertion fragments and two replacement fragments; 2) the NR gene of most inbred lines from Mo17 and derived lines had one insertion fragment and one replacement fragment; 3) there were two common variations and eight common SNPs in the NR gene of the four key lines. Huangzaosi and Mo17 also had three common variations compared with the other key lines. Moreover, Mo17 had some unique variations; there were no unique variations in the other key lines, even for SNPs, and 4) phylogenetic tree analysis showed that the NR gene of the derived lines from the same key line had higher sequence homology. Based on the above NR gene variation characteristics and sequence homology of key inbred lines and derived lines, a candidate inbred line can be preliminarily selected and evaluated by marker development and/or sequence alignment of the NR gene.

  15. Changing perspectives on the origin of eukaryotes.

    PubMed

    Katz, L A

    1998-12-01

    From the initial application of molecular techniques to the study of microbial organisms, three domains of life emerged, with eukaryotes and archaea as sister taxa. However, recent analyses of an expanding molecular data set reveal that the eukaryotic genome is chimeric with respect to archaea and bacteria. Moreover, there is now evidence that the primitive eukaryotic group `Archezoa' once harbored mitochondia. These discoveries have challenged the traditional stepwise model of the evolution of eukaryotes, in which the nucleus and microtubules evolve before the acquisition of mitochondria, and consequently compel a revision of existing models of the origin of eukaryotic cells. PMID:21238406

  16. Evolutionary position of breviate amoebae and the primary eukaryote divergence.

    PubMed

    Minge, Marianne A; Silberman, Jeffrey D; Orr, Russell J S; Cavalier-Smith, Thomas; Shalchian-Tabrizi, Kamran; Burki, Fabien; Skjaeveland, Asmund; Jakobsen, Kjetill S

    2009-02-22

    Integration of ultrastructural and molecular sequence data has revealed six supergroups of eukaryote organisms (excavates, Rhizaria, chromalveolates, Plantae, Amoebozoa and opisthokonts), and the root of the eukaryote evolutionary tree is suggested to lie between unikonts (Amoebozoa, opisthokonts) and bikonts (the other supergroups). However, some smaller lineages remain of uncertain affinity. One of these unassigned taxa is the anaerobic, free-living, amoeboid flagellate Breviata anathema, which is of key significance as it is unclear whether it is a unikont (i.e. possibly the deepest branching amoebozoan) or a bikont. To establish its evolutionary position, we sequenced thousands of Breviata genes and calculated trees using 78 protein sequences. Our trees and specific substitutions in the 18S RNA sequence indicate that Breviata is related to other Amoebozoa, thereby significantly increasing the cellular diversity of this phylum and establishing Breviata as a deep-branching unikont. We discuss the implications of these results for the ancestral state of Amoebozoa and eukaryotes generally, demonstrating that phylogenomics of phylogenetically 'nomadic' species can elucidate key questions in eukaryote evolution. Furthermore, mitochondrial genes among the Breviata ESTs demonstrate that Breviata probably contains a modified anaerobic mitochondrion. With these findings, remnants of mitochondria have been detected in all putatively deep-branching amitochondriate organisms.

  17. Genetics of eukaryotic RNA polymerases I, II, and III.

    PubMed Central

    Archambault, J; Friesen, J D

    1993-01-01

    The transcription of nucleus-encoded genes in eukaryotes is performed by three distinct RNA polymerases termed I, II, and III, each of which is a complex enzyme composed of more than 10 subunits. The isolation of genes encoding subunits of eukaryotic RNA polymerases from a wide spectrum of organisms has confirmed previous biochemical and immunological data indicating that all three enzymes are closely related in structures that have been conserved in evolution. Each RNA polymerase is an enzyme complex composed of two large subunits that are homologous to the two largest subunits of prokaryotic RNA polymerases and are associated with smaller polypeptides, some of which are common to two or to all three eukaryotic enzymes. This remarkable conservation of structure most probably underlies a conservation of function and emphasizes the likelihood that information gained from the study of RNA polymerases from one organism will be applicable to others. The recent isolation of many mutations affecting the structure and/or function of eukaryotic and prokaryotic RNA polymerases now makes it feasible to begin integrating genetic and biochemical information from various species in order to develop a picture of these enzymes. The picture of eukaryotic RNA polymerases depicted in this article emphasizes the role(s) of different polypeptide regions in interaction with other subunits, cofactors, substrates, inhibitors, or accessory transcription factors, as well as the requirement for these interactions in transcription initiation, elongation, pausing, termination, and/or enzyme assembly. Most mutations described here have been isolated in eukaryotic organisms that have well-developed experimental genetic systems as well as amenable biochemistry, such as Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. When relevant, mutations affecting regions of Escherichia coli RNA polymerase that are conserved among eukaryotes and prokaryotes are also presented

  18. Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes.

    PubMed

    Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

    2015-04-01

    Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

  19. Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes.

    PubMed

    Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

    2015-04-01

    Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance.

  20. Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes

    PubMed Central

    Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

    2015-01-01

    Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

  1. The dispersed archaeal eukaryome and the complex archaeal ancestor of eukaryotes.

    PubMed

    Koonin, Eugene V; Yutin, Natalya

    2014-04-01

    The ancestral set of eukaryotic genes is a chimera composed of genes of archaeal and bacterial origins thanks to the endosymbiosis event that gave rise to the mitochondria and apparently antedated the last common ancestor of the extant eukaryotes. The proto-mitochondrial endosymbiont is confidently identified as an α-proteobacterium. In contrast, the archaeal ancestor of eukaryotes remains elusive, although evidence is accumulating that it could have belonged to a deep lineage within the TACK (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) superphylum of the Archaea. Recent surveys of archaeal genomes show that the apparent ancestors of several key functional systems of eukaryotes, the components of the archaeal "eukaryome," such as ubiquitin signaling, RNA interference, and actin-based and tubulin-based cytoskeleton structures, are identifiable in different archaeal groups. We suggest that the archaeal ancestor of eukaryotes was a complex form, rooted deeply within the TACK superphylum, that already possessed some quintessential eukaryotic features, in particular, a cytoskeleton, and perhaps was capable of a primitive form of phagocytosis that would facilitate the engulfment of potential symbionts. This putative group of Archaea could have existed for a relatively short time before going extinct or undergoing genome streamlining, resulting in the dispersion of the eukaryome. This scenario might explain the difficulty with the identification of the archaeal ancestor of eukaryotes despite the straightforward detection of apparent ancestors to many signature eukaryotic functional systems.

  2. The Dispersed Archaeal Eukaryome and the Complex Archaeal Ancestor of Eukaryotes

    PubMed Central

    Koonin, Eugene V.; Yutin, Natalya

    2014-01-01

    The ancestral set of eukaryotic genes is a chimera composed of genes of archaeal and bacterial origins thanks to the endosymbiosis event that gave rise to the mitochondria and apparently antedated the last common ancestor of the extant eukaryotes. The proto-mitochondrial endosymbiont is confidently identified as an α-proteobacterium. In contrast, the archaeal ancestor of eukaryotes remains elusive, although evidence is accumulating that it could have belonged to a deep lineage within the TACK (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) superphylum of the Archaea. Recent surveys of archaeal genomes show that the apparent ancestors of several key functional systems of eukaryotes, the components of the archaeal “eukaryome,” such as ubiquitin signaling, RNA interference, and actin-based and tubulin-based cytoskeleton structures, are identifiable in different archaeal groups. We suggest that the archaeal ancestor of eukaryotes was a complex form, rooted deeply within the TACK superphylum, that already possessed some quintessential eukaryotic features, in particular, a cytoskeleton, and perhaps was capable of a primitive form of phagocytosis that would facilitate the engulfment of potential symbionts. This putative group of Archaea could have existed for a relatively short time before going extinct or undergoing genome streamlining, resulting in the dispersion of the eukaryome. This scenario might explain the difficulty with the identification of the archaeal ancestor of eukaryotes despite the straightforward detection of apparent ancestors to many signature eukaryotic functional systems. PMID:24691961

  3. Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells

    PubMed Central

    Guzmán-Pérez, Valentina; Bumke-Vogt, Christiane; Schreiner, Monika; Mewis, Inga; Borchert, Andrea; Pfeiffer, Andreas F. H.

    2016-01-01

    Nasturtium (Tropaeolum majus L.) contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate—the benzyl isothiocyanate (BITC)—modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1). FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC—extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase—to modulate i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and, iii) the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB) and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived)-like2 (NRF2) and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance. PMID:27622707

  4. Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

    PubMed

    Guzmán-Pérez, Valentina; Bumke-Vogt, Christiane; Schreiner, Monika; Mewis, Inga; Borchert, Andrea; Pfeiffer, Andreas F H

    2016-01-01

    Nasturtium (Tropaeolum majus L.) contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC)-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1). FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and, iii) the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB) and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived)-like2 (NRF2) and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance. PMID:27622707

  5. Brain-derived neurotrophic factor gene-modified bone marrow mesenchymal stem cells

    PubMed Central

    HAN, ZHONG-MIN; HUANG, HE-MEI; WANG, FEI-FEI

    2015-01-01

    The present study aimed to investigate the effects of human brain-derived neurotrophic factor (hBDNF) on the differentiation of bone marrow mesenchymal stem cells (MSCs) into neuron-like cells. Lentiviral vectors carrying the hBDNF gene were used to modify the bone marrow stromal cells (BMSCs) of Sprague-Dawley (SD) rats. The rat BMSCs were isolated, cultured and identified. A lentivirus bearing hBDNF and enhanced green fluorescent protein (eGFP) genes was subcultured and used to infect the SD rat BMSCs. The expression of eGFP was observed under a fluorescence microscope to determine the infection rate and growth of the transfected cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect the proliferation rate of cells following transfection. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to detect the expression levels of hBDNF. Differentiation of neuron-like cells was induced in vitro and the differentiation rate of the induced neural-like cells was compared with that in control groups and analyzed statistically. In the cultured cells, flow cytometry demonstrated positive expression of cluster of differentiation (CD)90 and CD44, and negative expression of CD34 and CD45. The proliferation rate of the rat BMSCs increased following gene transfection. The expression of hBDNF-eGFP was detected in the BMSCs of the experimental group. The differentiation rate of hBDNF-modified cells into neuron-like cells in the experimental group was higher compared with that in empty plasmid and untransfected negative control groups. The difference was statistically significant (P<0.05). Thus, BDNF gene transfection is able to promote the differentiation of BMSCs into neuron-like cells. BDNF may play an important role in the differentiation of MSCs into neuron-like cells. PMID:25574226