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Sample records for automated eukaryotic gene

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Amplification and characterization of eukaryotic structural genes.

    Science.gov (United States)

    Maniatis, T; Efstratiadis, A; Sim, G K; Kafatos, F

    1978-05-01

    An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.

  3. Chlamydial genes shed light on the evolution of photoautotrophic eukaryotes

    OpenAIRE

    2008-01-01

    Abstract Background Chlamydiae are obligate intracellular bacteria of protists, invertebrates and vertebrates, but have not been found to date in photosynthetic eukaryotes (algae and embryophytes). Genes of putative chlamydial origin, however, are present in significant numbers in sequenced genomes of photosynthetic eukaryotes. It has been suggested that such genes were acquired by an ancient horizontal gene transfer from Chlamydiae to the ancestor of photosynthetic eukaryotes. To further tes...

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

    OpenAIRE

    Thiergart, T.; Landan, G; Schenk, M.; Dagan, T.; Martin, W 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 a...

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

    Science.gov (United States)

    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.

  6. Rooting the eukaryote tree by using a derived gene fusion.

    Science.gov (United States)

    Stechmann, Alexandra; Cavalier-Smith, Thomas

    2002-07-05

    Single-gene trees have failed to locate the root of the eukaryote tree because of systematic biases in sequence evolution. Structural genetic data should yield more reliable insights into deep phylogenetic relationships. We searched major protist groups for the presence or absence of a gene fusion in order to locate the root of the eukaryote tree. In striking contrast to previous molecular studies, we show that all eukaryote groups ancestrally with two cilia (bikonts) are evolutionarily derived. The root lies between bikonts and opisthokonts (animals, Fungi, Choanozoa). Amoebozoa either diverged even earlier or are sister of bikonts or (less likely) opisthokonts.

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

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    Zhao Sen

    2012-09-01

    Full Text Available Abstract 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.

  8. Chlamydial genes shed light on the evolution of photoautotrophic eukaryotes

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    Melkonian Michael

    2008-07-01

    Full Text Available Abstract Background Chlamydiae are obligate intracellular bacteria of protists, invertebrates and vertebrates, but have not been found to date in photosynthetic eukaryotes (algae and embryophytes. Genes of putative chlamydial origin, however, are present in significant numbers in sequenced genomes of photosynthetic eukaryotes. It has been suggested that such genes were acquired by an ancient horizontal gene transfer from Chlamydiae to the ancestor of photosynthetic eukaryotes. To further test this hypothesis, an extensive search for proteins of chlamydial origin was performed using several recently sequenced algal genomes and EST databases, and the proteins subjected to phylogenetic analyses. Results A total of 39 proteins of chlamydial origin were retrieved from the photosynthetic eukaryotes analyzed and their identity verified through phylogenetic analyses. The distribution of the chlamydial proteins among four groups of photosynthetic eukaryotes (Viridiplantae, Rhodoplantae, Glaucoplantae, Bacillariophyta was complex suggesting multiple acquisitions and losses. Evidence is presented that all except one of the chlamydial genes originated from an ancient endosymbiosis of a chlamydial bacterium into the ancestor of the Plantae before their divergence into Viridiplantae, Rhodoplantae and Glaucoplantae, i.e. more than 1.1 BYA. The chlamydial proteins subsequently spread through secondary plastid endosymbioses to other eukaryotes. Of 20 chlamydial proteins recovered from the genomes of two Bacillariophyta, 10 were of rhodoplant, and 10 of viridiplant origin suggesting that they were acquired by two different secondary endosymbioses. Phylogenetic analyses of concatenated sequences demonstrated that the viridiplant secondary endosymbiosis likely occurred before the divergence of Chlorophyta and Streptophyta. Conclusion We identified 39 proteins of chlamydial origin in photosynthetic eukaryotes signaling an ancient invasion of the ancestor of the

  9. Horizontal gene transfer in the evolution of photosynthetic eukaryotes

    Institute of Scientific and Technical Information of China (English)

    Jinling HUANG; Jipei YUE

    2013-01-01

    Horizontal gene transfer (HGT) may not only create genome mosaicism,but also introduce evolutionary novelties to recipient organisms.HGT in plastid genomes,though relatively rare,still exists.HGT-derived genes are particularly common in unicellular photosynthetic eukaryotes and they also occur in multicellular plants.In particular,ancient HGT events occurring during the early evolution of primary photosynthetic eukaryotes were probably frequent.There is clear evidence that anciently acquired genes played an important role in the establishment of primary plastids and in the transition of plants from aquatic to terrestrial environments.Although algal genes have often been used to infer historical plastids in plastid-lacking eukaryotes,reliable approaches are needed to distinguish endosymbionts-derived genes from those independently acquired from preferential feeding or other activities.

  10. An evolutionary network of genes present in the eukaryote common ancestor polls genomes on eukaryotic and mitochondrial origin.

    Science.gov (United States)

    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 dialect

  11. Automatic generation of gene finders for eukaryotic species

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Krogh, A.

    2006-01-01

    Background The number of sequenced eukaryotic genomes is rapidly increasing. This means that over time it will be hard to keep supplying customised gene finders for each genome. This calls for procedures to automatically generate species-specific gene finders and to re-train them as the quantity...

  12. Distinct gene number-genome size relationships for eukaryotes and non-eukaryotes: gene content estimation for dinoflagellate genomes.

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    Yubo Hou

    Full Text Available The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log(10-transformed protein-coding gene number (Y' versus log(10-transformed genome size (X', genome size in kbp were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Y' = ln(-46.200+22.678X', whereas non-eukaryotes a linear model, Y' = 0.045+0.977X', both with high significance (p0.91. Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%-1% compared to higher and relatively stable percentages in prokaryotes and viruses (97%-47%. The eukaryotic regression models project that the smallest dinoflagellate genome (3x10(6 kbp contains 38,188 protein-coding (40,086 total genes and the largest (245x10(6 kbp 87,688 protein-coding (92,013 total genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species.

  13. Noise minimization in eukaryotic gene expression.

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    Hunter B Fraser

    2004-06-01

    Full Text Available 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.

  14. Eukaryote-to-eukaryote gene transfer gives rise to genome mosaicism in euglenids

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    Weber Andreas PM

    2011-04-01

    Full Text Available Abstract Background Euglenophytes are a group of photosynthetic flagellates possessing a plastid derived from a green algal endosymbiont, which was incorporated into an ancestral host cell via secondary endosymbiosis. However, the impact of endosymbiosis on the euglenophyte nuclear genome is not fully understood due to its complex nature as a 'hybrid' of a non-photosynthetic host cell and a secondary endosymbiont. Results We analyzed an EST dataset of the model euglenophyte Euglena gracilis using a gene mining program designed to detect laterally transferred genes. We found E. gracilis genes showing affinity not only with green algae, from which the secondary plastid in euglenophytes evolved, but also red algae and/or secondary algae containing red algal-derived plastids. Phylogenetic analyses of these 'red lineage' genes suggest that E. gracilis acquired at least 14 genes via eukaryote-to-eukaryote lateral gene transfer from algal sources other than the green algal endosymbiont that gave rise to its current plastid. We constructed an EST library of the aplastidic euglenid Peranema trichophorum, which is a eukaryovorous relative of euglenophytes, and also identified 'red lineage' genes in its genome. Conclusions Our data show genome mosaicism in E. gracilis and P. trichophorum. One possible explanation for the presence of these genes in these organisms is that some or all of them were independently acquired by lateral gene transfer and contributed to the successful integration and functioning of the green algal endosymbiont as a secondary plastid. Alternative hypotheses include the presence of a phagocytosed alga as the single source of those genes, or a cryptic tertiary endosymbiont harboring secondary plastid of red algal origin, which the eukaryovorous ancestor of euglenophytes had acquired prior to the secondary endosymbiosis of a green alga.

  15. Chromatin—a global buffer for eukaryotic gene control

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    Yuri M. Moshkin

    2015-09-01

    Full Text Available Most of eukaryotic DNA is embedded into nucleosome arrays formed by DNA wrapped around a core histone octamer. Nucleosome is a fundamental repeating unit of chromatin guarding access to the genetic information. Here, I will discuss two facets of nucleosome in eukaryotic gene control. On the one hand, nucleosome acts as a regulatory unit, which controls gene switches through a set of post-translational modifications occurring on histone tails. On the other hand, global configuration of nucleosome arrays with respect to nucleosome positioning, spacing and turnover acts as a tuning parameter for all genomic functions. A “histone code” hypothesis extents the Jacob-Monod model for eukaryotic gene control; however, when considering factors capable of reconfiguring entire nucleosome array, such as ATP-dependent chromatin remodelers, this model becomes limited. Global changes in nucleosome arrays will be sensed by every gene, yet the transcriptional responses might be specific and appear as gene targeted events. What determines such specificity is unclear, but it’s likely to depend on initial gene settings, such as availability of transcription factors, and on configuration of new nucleosome array state.

  16. The effect of negative autoregulation on eukaryotic gene expression

    Science.gov (United States)

    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.

  17. Evolution of filamentous plant pathogens: gene exchange across eukaryotic kingdoms.

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    Richards, Thomas A; Dacks, Joel B; Jenkinson, Joanna M; Thornton, Christopher R; Talbot, Nicholas J

    2006-09-19

    Filamentous fungi and oomycetes are eukaryotic microorganisms that grow by producing networks of thread-like hyphae, which secrete enzymes to break down complex nutrients, such as wood and plant material, and recover the resulting simple sugars and amino acids by osmotrophy. These organisms are extremely similar in both appearance and lifestyle and include some of the most economically important plant pathogens . However, the morphological similarity of fungi and oomycetes is misleading because they represent some of the most distantly related eukaryote evolutionary groupings, and their shared osmotrophic growth habit is interpreted as being the result of convergent evolution . The fungi branch with the animals, whereas the oomycetes branch with photosynthetic algae as part of the Chromalveolata . In this report, we provide strong phylogenetic evidence that multiple horizontal gene transfers (HGT) have occurred from filamentous ascomycete fungi to the distantly related oomycetes. We also present evidence that a subset of the associated gene families was initially the product of prokaryote-to-fungi HGT. The predicted functions of the gene products associated with fungi-to-oomycete HGT suggest that this process has played a significant role in the evolution of the osmotrophic, filamentous lifestyle on two separate branches of the eukaryote tree.

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

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    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.

  19. Methods for identifying and mapping recent segmental and gene duplications in eukaryotic genomes.

    Science.gov (United States)

    Khaja, Razi; MacDonald, Jeffrey R; Zhang, Junjun; Scherer, Stephen W

    2006-01-01

    The aim of this chapter is to provide instruction for analyzing and mapping recent segmental and gene duplications in eukaryotic genomes. We describe a bioinformatics-based approach utilizing computational tools to manage eukaryotic genome sequences to characterize and understand the evolutionary fates and trajectories of duplicated genes. An introduction to bioinformatics tools and programs such as BLAST, Perl, BioPerl, and the GFF specification provides the necessary background to complete this analysis for any eukaryotic genome of interest.

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

    Science.gov (United States)

    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).

  1. Widespread Inter- and Intra-Domain Horizontal Gene Transfer of d-Amino Acid Metabolism Enzymes in Eukaryotes

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    Naranjo-Ortíz, Miguel A.; Brock, Matthias; Brunke, Sascha; Hube, Bernhard; Marcet-Houben, Marina; Gabaldón, Toni

    2016-01-01

    Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that d-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether d-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role. PMID:28066338

  2. Expression of Exogenous Gene in Prokaryotes and Eukaryotes

    Institute of Scientific and Technical Information of China (English)

    雷正玉; 张晓

    2014-01-01

    [目的]研究外源基因在原核生物和真核生物中的表达。[方法]将带有目的基因的表达载体 pTYB2-WF转化大肠杆菌 ER2566,用 IPTG诱导植酸酶基因表达。用 SDS-PAGE验证植酸酶融合蛋白表达情况,并进一步对融合蛋白进行纯化。用ç赤酵母表达系统表达植酸酶基因 phyA;构建酵母整合载体pPIC9K-phyA,转化ç赤酵母 GS115,构建工程菌 GS115-pPIC9K-phyA。[结果]在甲醇的诱导下表达植酸酶,经酶活力的测定,其植酸酶活力达7.3μ/ml,构建了ç赤酵母工程菌GS115-pPIC9K-phyA。[结论]甲醇酵母表达机制在分子生物学以及工业应用领域发挥作用。%Objective This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.

  3. Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria

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    Keeling Patrick J

    2007-06-01

    Full Text Available Abstract Background Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes. Results Here, we show that abundant marine cyanobacteria in the related genera Synechococcus and Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA are characteristic of eukaryotes and prokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream of the ancestral prokaryotic gene in several strains (ecotypes of Synechococcus and Prochlorococcus. In one lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is most closely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replaced the plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functional advantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scattered across the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferred multiple times among cyanobacteria, or more likely because it has been selectively retained only in certain lineages. Conclusion A gene for plastid-targeted FBA has been transferred from red algae to cyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Its current distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex history since its introduction to this group.

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

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    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.

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

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    Kenan Hadziavdic

    Full Text Available 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.

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

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    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.

  7. Algal endosymbionts as vectors of horizontal gene transfer in photosynthetic eukaryotes

    Directory of Open Access Journals (Sweden)

    Huan eQiu

    2013-09-01

    Full Text Available Photosynthesis in eukaryotes occurs in the plastid, an organelle that is derived from a single cyanobacterial primary endosymbiosis in the common ancestor of the supergroup Plantae (or Archaeplastida that includes green, red, and glaucophyte algae and plants. However a variety of other phytoplankton such as the chlorophyll c-containing diatoms, dinoflagellates, and haptophytes contain a red alga-derived plastid that traces its origin to secondary or tertiary (eukaryote engulfs eukaryote endosymbiosis. The hypothesis of Plantae monophyly has only recently been substantiated, however the extent and role of endosymbiotic and horizontal gene transfer (EGT and HGT in algal genome evolution still remain to be fully understood. What is becoming clear from analysis of complete genome data is that algal gene complements can no longer be considered essentially eukaryotic in provenance; i.e., with the expected addition of several hundred cyanobacterial genes derived from EGT and a similar number derived from the mitochondrial ancestor. For example, we now know that foreign cells such as Chlamydiae and other prokaryotes have made significant contributions to plastid functions in Plantae. Perhaps more surprising is the recent finding of extensive bacterium-derived HGT in the nuclear genome of the unicellular red alga Porphyridium purpureum that does not relate to plastid functions. These non-endosymbiont gene transfers not only shaped the evolutionary history of Plantae but also were propagated via secondary endosymbiosis to a multitude of other phytoplankton. Here we discuss the idea that Plantae (in particular red algae are one of the major players in eukaryote genome evolution by virtue of their ability to act as sinks and sources of foreign genes through HGT and endosymbiosis, respectively. This hypothesis recognizes the often under-appreciated Rhodophyta as major sources of genetic novelty among photosynthetic eukaryotes.

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

    Science.gov (United States)

    Li, Ting; Huang, Sheng; Zhao, Xuefeng; Wright, David A; Carpenter, Susan; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-08-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes.

    Science.gov (United States)

    Matys, V; Kel-Margoulis, O V; Fricke, E; Liebich, I; Land, S; Barre-Dirrie, A; Reuter, I; Chekmenev, D; Krull, M; Hornischer, K; Voss, N; Stegmaier, P; Lewicki-Potapov, B; Saxel, H; Kel, A E; Wingender, E

    2006-01-01

    The TRANSFAC database on transcription factors, their binding sites, nucleotide distribution matrices and regulated genes as well as the complementing database TRANSCompel on composite elements have been further enhanced on various levels. A new web interface with different search options and integrated versions of Match and Patch provides increased functionality for TRANSFAC. The list of databases which are linked to the common GENE table of TRANSFAC and TRANSCompel has been extended by: Ensembl, UniGene, EntrezGene, HumanPSD and TRANSPRO. Standard gene names from HGNC, MGI and RGD, are included for human, mouse and rat genes, respectively. With the help of InterProScan, Pfam, SMART and PROSITE domains are assigned automatically to the protein sequences of the transcription factors. TRANSCompel contains now, in addition to the COMPEL table, a separate table for detailed information on the experimental EVIDENCE on which the composite elements are based. Finally, for TRANSFAC, in respect of data growth, in particular the gain of Drosophila transcription factor binding sites (by courtesy of the Drosophila DNase I footprint database) and of Arabidopsis factors (by courtesy of DATF, Database of Arabidopsis Transcription Factors) has to be stressed. The here described public releases, TRANSFAC 7.0 and TRANSCompel 7.0, are accessible under http://www.gene-regulation.com/pub/databases.html.

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

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    Xie Jiatao

    2011-09-01

    Full Text Available Abstract 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.

  12. Correlating overrepresented upstream motifs to gene expression a computational approach to regulatory element discovery in eukaryotes

    CERN Document Server

    Caselle, M; Provero, P

    2002-01-01

    Gene regulation in eukaryotes is mainly effected through transcription factors binding to rather short recognition motifs generally located upstream of the coding region. We present a novel computational method to identify regulatory elements in the upstream region of eukaryotic genes. The genes are grouped in sets sharing an overrepresented short motif in their upstream sequence. For each set, the average expression level from a microarray experiment is determined: If this level is significantly higher or lower than the average taken over the whole genome, then the overerpresented motif shared by the genes in the set is likely to play a role in their regulation. The method was tested by applying it to the genome of Saccharomyces cerevisiae, using the publicly available results of a DNA microarray experiment, in which expression levels for virtually all the genes were measured during the diauxic shift from fermentation to respiration. Several known motifs were correctly identified, and a new candidate regulat...

  13. Balancing noise and plasticity in eukaryotic gene expression

    Directory of Open Access Journals (Sweden)

    Bajić Djordje

    2012-07-01

    Full Text Available Abstract Background Coupling the control of expression stochasticity (noise to the ability of expression change (plasticity can alter gene function and influence adaptation. A number of factors, such as transcription re-initiation, strong chromatin regulation or genome neighboring organization, underlie this coupling. However, these factors do not necessarily combine in equivalent ways and strengths in all genes. Can we identify then alternative architectures that modulate in distinct ways the linkage of noise and plasticity? Results Here we first show that strong chromatin regulation, commonly viewed as a source of coupling, can lead to plasticity without noise. The nature of this regulation is relevant too, with plastic but noiseless genes being subjected to general activators whereas plastic and noisy genes experience more specific repression. Contrarily, in genes exhibiting poor transcriptional control, it is translational efficiency what separates noise from plasticity, a pattern related to transcript length. This additionally implies that genome neighboring organization –as modifier– appears only effective in highly plastic genes. In this class, we confirm bidirectional promoters (bipromoters as a configuration capable to reduce coupling by abating noise but also reveal an important trade-off, since bipromoters also decrease plasticity. This presents ultimately a paradox between intergenic distances and modulation, with short intergenic distances both associated and disassociated to noise at different plasticity levels. Conclusions Balancing the coupling among different types of expression variability appears as a potential shaping force of genome regulation and organization. This is reflected in the use of different control strategies at genes with different sets of functional constraints.

  14. Construction and Expression of Eukaryotic Expression Vector of Mature Polypeptide of Duck Interferon Alpha Gene

    Institute of Scientific and Technical Information of China (English)

    PEI Fucheng; LI Jingpeng; LI Lu; ZHANG Jianguang; REN Guiping

    2006-01-01

    To study biological activities of Duck Interferon Alpha (DuIFN-α) and prepare antivirus medicine, the eukaryotic expression vector of mature polypeptide of Duck Interferon Alpha (mDuIFN-α) gene was constructed and expressed in insect cell. By means of PCR technique, the mDuIFN-α gene was cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. The mDuIFN-α gene was ligated with the eukaryotic expression vector pMelBacA, then transfected into Sf9cell line. Recombinant polypeptide was effectively expressed in insect cell and its molecular weight was 34 ku.

  15. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

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

    Science.gov (United States)

    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.

  17. Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

    Directory of Open Access Journals (Sweden)

    Butler Margaret I

    2006-10-01

    Full Text Available Abstract Background Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. Results We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2 from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2 of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2, one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. Conclusion The identification of these new inteins

  18. Peptides encoded by short ORFs control development and define a new eukaryotic gene family.

    Directory of Open Access Journals (Sweden)

    Máximo Ibo Galindo

    2007-05-01

    Full Text Available Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal, a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs, which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.

  19. Construction and Identification of Human Tissue Kallikrein Gene Eukaryotic Expressing Vector

    Institute of Scientific and Technical Information of China (English)

    DAI Yong; PENG Wujian; LI Tiyuan; DU Hong; SUN Wenxue; CHEN Deheng; XU Zhuojia

    2007-01-01

    To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT)primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer.Then the KKgene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy...

  20. Snapshot of the eukaryotic gene expression in muskoxen rumen--a metatranscriptomic approach.

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    Meng Qi

    Full Text Available 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.

  1. [Establishment of a novel biotin-inducible eukaryotic gene regulation system].

    Science.gov (United States)

    Ye, Lingling; Hong, Liu; Li, Shichong; Wang, Qiwei; Lan, Sanchun; Chen, Zhaolie

    2014-08-01

    To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.

  2. WebAUGUSTUS--a web service for training AUGUSTUS and predicting genes in eukaryotes.

    Science.gov (United States)

    Hoff, Katharina J; Stanke, Mario

    2013-07-01

    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.

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

    Science.gov (United States)

    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

  4. Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms.

    Science.gov (United States)

    Xu, Lin; Chen, Hong; Hu, Xiaohua; Zhang, Rongmei; Zhang, Ze; Luo, Z W

    2006-06-01

    The average length of genes in a eukaryote is larger than in a prokaryote, implying that evolution of complexity is related to change of gene lengths. Here, we show that although the average lengths of genes in prokaryotes and eukaryotes are much different, the average lengths of genes are highly conserved within either of the two kingdoms. This suggests that natural selection has clearly set a strong limitation on gene elongation within the kingdom. Furthermore, the average gene size adds another distinct characteristic for the discrimination between the two kingdoms of organisms.

  5. New organelles by gene duplication in a biophysical model of eukaryote endomembrane evolution.

    Science.gov (United States)

    Ramadas, Rohini; Thattai, Mukund

    2013-06-04

    Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification.

  6. Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Tao; JIANG Xiao-dan; XU Zhong; YUAN Jun; DING Lian-shu; ZOU Yu-xi; XU Ru-xiang

    2005-01-01

    Objective: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. Methods: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. Results: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. Conclusions: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  11. Lateral transfer of tetrahymanol-synthesizing genes has allowed multiple diverse eukaryote lineages to independently adapt to environments without oxygen

    Directory of Open Access Journals (Sweden)

    Takishita Kiyotaka

    2012-02-01

    Full Text Available Abstract Sterols are key components of eukaryotic cellular membranes that are synthesized by multi-enzyme pathways that require molecular oxygen. Because prokaryotes fundamentally lack sterols, it is unclear how the vast diversity of bacterivorous eukaryotes that inhabit hypoxic environments obtain, or synthesize, sterols. Here we show that tetrahymanol, a triterpenoid that does not require molecular oxygen for its biosynthesis, likely functions as a surrogate of sterol in eukaryotes inhabiting oxygen-poor environments. Genes encoding the tetrahymanol synthesizing enzyme squalene-tetrahymanol cyclase were found from several phylogenetically diverged eukaryotes that live in oxygen-poor environments and appear to have been laterally transferred among such eukaryotes. Reviewers This article was reviewed by Eric Bapteste and Eugene Koonin.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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).

  14. Preferential duplication of intermodular hub genes: an evolutionary signature in eukaryotes genome networks.

    Directory of Open Access Journals (Sweden)

    Ricardo M Ferreira

    Full Text Available Whole genome protein-protein association networks are not random and their topological properties stem from genome evolution mechanisms. In fact, more connected, but less clustered proteins are related to genes that, in general, present more paralogs as compared to other genes, indicating frequent previous gene duplication episodes. On the other hand, genes related to conserved biological functions present few or no paralogs and yield proteins that are highly connected and clustered. These general network characteristics must have an evolutionary explanation. Considering data from STRING database, we present here experimental evidence that, more than not being scale free, protein degree distributions of organisms present an increased probability for high degree nodes. Furthermore, based on this experimental evidence, we propose a simulation model for genome evolution, where genes in a network are either acquired de novo using a preferential attachment rule, or duplicated with a probability that linearly grows with gene degree and decreases with its clustering coefficient. For the first time a model yields results that simultaneously describe different topological distributions. Also, this model correctly predicts that, to produce protein-protein association networks with number of links and number of nodes in the observed range for Eukaryotes, it is necessary 90% of gene duplication and 10% of de novo gene acquisition. This scenario implies a universal mechanism for genome evolution.

  15. Establishment of the methods for searching eukaryotic gene cis-regulatory modules.

    Science.gov (United States)

    Zhong, Dong; Zhang, Zhen-shu; Liu, Yu-hu; Zheng, Guo-qing; Liu, Xiao-yi; Lu, Yang; Zhao, Gui-jun; Xu, An-long

    2004-02-01

    On the basis of the knowledge of eukaryotic gene regulation, we modified the method in three aspects: (1) Searching the cis-regulatory modules (CRM) according Fasta or Blast sequence with multiple sequence and low E value, followed by mutual scoring of these sequence with Smith-Waterman algorithms and finally by clustering analysis; (2) Searching the transcription factor-binding site using International Union of Pure and Applied Chemistry, Position-Weight Matrix(PWM) and Dyed method; (3) Designing and implementation of data analysis based on the software in Windows 2000 and UNIX using object-oriented technology. The results of analysis of the major histocompatibility complex gene family show that this procedure may accurately locate the regions that contain some of the CRMs.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Coralie Damon

    Full Text Available 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

  19. [Expression of target gene in eukaryotic cells driven by prokaryotic T7 promoter and its RNA polymerase].

    Science.gov (United States)

    Yuan, Zhi-Gang; Zhang, Jin-Ping; Chu, Yi-Wei; Wang, Ying; Xu, Wei; Xiong, Si-Dong

    2005-03-01

    To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.

  20. CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

  1. Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

    Directory of Open Access Journals (Sweden)

    Fournier Pierre-Edouard

    2009-03-01

    Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

  2. The Eukaryotic DNMT2 Genes Encode a New Class of Cytosine-5 DNA Methyltransferases

    Institute of Scientific and Technical Information of China (English)

    Lin-YaTang; M.NarsaReddy; VanyaRasheva; Tai-LinLee; Meng-JauLin; Ming-ShiuHung; C.-K.JamesShen

    2005-01-01

    DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have showm that thegenome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anoma-lously methylated at the 5'-positions of cytosines(Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J.(2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mousc ortholog, mDnmt2, are capable of methylating a cotrans-fected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.

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

    Science.gov (United States)

    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.

  4. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  5. Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Ya-pei; XU Chun-xiao; HOU Guo-sheng; XIN Jia-xuan; WANG Wei; LIU Xian-xi

    2010-01-01

    Background Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein.The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.Methods The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN.Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting.Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry.To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.Results DNA sequence confirmed that pcDNA-tumstatin was successfully constructed.RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively.After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase.And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.Conclusions Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.

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

    OpenAIRE

    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 ...

  7. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Shan Qin; Hong Tang; Lian-San Zhao; Fang He; Yong Lin; Li Liu; Xiao-Mei He

    2003-01-01

    AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV, or BamHI/EcoR Ⅰ followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSGSUTPL/Flag plasmid vectors with T4DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preSl-preS2S and preSlS encoding genes,determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins.Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.CONCLUSION: Eight recombinant plasmids expressing S,M, L or preSlS proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Michel, Christian J

    2015-09-07

    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

  11. Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba Ⅰ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP Ⅱ for Uroplakin Ⅱ was successfully constructed.After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

  12. Eukaryotic Expression of Human Arresten Gene and Its Effect on the Proliferation of Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    SHANG Dan; ZHENG Qichang; SONG Zifang; LI Yiqing; WANG Xiedan; GUO Xingjun

    2006-01-01

    The eukaryotic expression of human arresten geneand its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells,while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-αactin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P<0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

  13. Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates

    Science.gov (United States)

    Inupakutika, Madhuri A.; Sengupta, Soham; Nechushtai, Rachel; Jennings, Patricia A.; Onuchic, Jose’ N.; Azad, Rajeev K.; Padilla, Pamela; Mittler, Ron

    2017-02-01

    NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins.

  14. Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

    Institute of Scientific and Technical Information of China (English)

    FENG Yong; ZHENG Dong; YANG Shuhua; LI Jing

    2007-01-01

    The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supematants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Marissa B Hirst

    Full Text Available 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

  17. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    Science.gov (United States)

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  18. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David;

    2007-01-01

    Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Luisa W Hugerth

    Full Text Available 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.

  1. Cloning of Human α-defensin-1 (HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    Hua-Hua CHEN; Jing-Ping Ou YANG; Bao-Hua WANG; Yue Yang; Han-Qiao ZHENG

    2005-01-01

    @@ 1 Introduction Defensins are small cationic antimicrobial peptides that function in the host's innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3,which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidative mechanism in macrophages, and can direct inactivate the enveloped viruses. Because only special cells express defensins. And it is hard to extract them naturally and the production is few. So researcher expect to obtain defensins highly through heterogenous expression by gene engineering technology. In order to express HNP-1, we cloned the cDNA of HNP-1 from human polymorphonuclear cells in peripheral blood, and constructed its eukaryotic expression vector, which provided a base for the further study on its mechanism of antimicrobial effect.

  2. Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

    Science.gov (United States)

    Alekseev, Sergey; Nagy, Zita; Sandoz, Jérémy; Weiss, Amélie; Egly, Jean-Marc; Le May, Nicolas; Coin, Frederic

    2017-02-02

    Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.

  3. GOPET: A tool for automated predictions of Gene Ontology terms

    Directory of Open Access Journals (Sweden)

    Glatting Karl-Heinz

    2006-03-01

    Full Text Available Abstract Background Vast progress in sequencing projects has called for annotation on a large scale. A Number of methods have been developed to address this challenging task. These methods, however, either apply to specific subsets, or their predictions are not formalised, or they do not provide precise confidence values for their predictions. Description We recently established a learning system for automated annotation, trained with a broad variety of different organisms to predict the standardised annotation terms from Gene Ontology (GO. Now, this method has been made available to the public via our web-service GOPET (Gene Ontology term Prediction and Evaluation Tool. It supplies annotation for sequences of any organism. For each predicted term an appropriate confidence value is provided. The basic method had been developed for predicting molecular function GO-terms. It is now expanded to predict biological process terms. This web service is available via http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar Conclusion Our web service gives experimental researchers as well as the bioinformatics community a valuable sequence annotation device. Additionally, GOPET also provides less significant annotation data which may serve as an extended discovery platform for the user.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, A L [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    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.

  6. Automated alignment-based curation of gene models in filamentous fungi

    OpenAIRE

    2014-01-01

    Background Automated gene-calling is still an error-prone process, particularly for the highly plastic genomes of fungal species. Improvement through quality control and manual curation of gene models is a time-consuming process that requires skilled biologists and is only marginally performed. The wealth of available fungal genomes has not yet been exploited by an automated method that applies quality control of gene models in order to obtain more accurate genome annotations. Results We prov...

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

    Science.gov (United States)

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

    Full Text Available 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. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε 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 βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H 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 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N 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.

  11. Biochemical examination of the potential eukaryotic-type protein kinase genes in the complete genome of the unicellular Cyanobacterium synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kamei, Ayako; Yuasa, Takashi; Geng, Xiaoxing; Ikeuchi, Masahiko

    2002-06-30

    The complete genome of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for a subfamily Pkn2 of the eukaryotic-type (or "Hanks-type") protein kinase. Previously, SpkA and SpkB were shown to have protein kinase activity and to be required for cell motility. Here, the other five genes were examined. These genes, except for spkG (slr0152), were successfully expressed in Escherichia coli. Eukaryotic-type protein kinase activity of the expressed SpkC (Slr0599), SpkD (S110776) and SpkF (Slr1225) was demonstrated as autophosphorylation and phosphorylation of the general substrate proteins. SpkE (Slr1443) did not show any activity, a finding consistent with its lack of several key amino acid residues in its kinase motif. Gene-disrupted mutants showed no discernible defect in phenotype except that spkD was apparently essential for survival.

  12. 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.

    Science.gov (United States)

    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.

  13. Interactome and Gene Ontology provide congruent yet subtly different views of a eukaryotic cell

    Directory of Open Access Journals (Sweden)

    Marín Ignacio

    2009-07-01

    Full Text Available Abstract Background The characterization of the global functional structure of a cell is a major goal in bioinformatics and systems biology. Gene Ontology (GO and the protein-protein interaction network offer alternative views of that structure. Results This study presents a comparison of the global structures of the Gene Ontology and the interactome of Saccharomyces cerevisiae. Sensitive, unsupervised methods of clustering applied to a large fraction of the proteome led to establish a GO-interactome correlation value of +0.47 for a general dataset that contains both high and low-confidence interactions and +0.58 for a smaller, high-confidence dataset. Conclusion The structures of the yeast cell deduced from GO and interactome are substantially congruent. However, some significant differences were also detected, which may contribute to a better understanding of cell function and also to a refinement of the current ontologies.

  14. Predicting Polymerase Ⅱ Core Promoters by Cooperating Transcription Factor Binding Sites in Eukaryotic Genes

    Institute of Scientific and Technical Information of China (English)

    Xiao-Tu MA; Min-Ping QIAN; Hai-Xu TANG

    2004-01-01

    Several discriminate functions for predicting core promoters that based on the potential cooperation between transcription factor binding sites (TFBSs) are discussed. It is demonstrated that the promoter predicting accuracy is improved when the cooperation among TFBSs is taken into consideration.The core promoter region of a newly discovered gene CKLFSF1 is predicted to locate more than 1.5 kb far away from the 5′ end of the transcript and in the last intron of its upstream gene, which is experimentally confirmed later. The core promoters of 3402 human RefSeq sequences, obtained by extending the mRNAs in human genome sequences, are predicted by our algorithm, and there are about 60% of the predicted core promoters locating within the ± 500 bp region relative to the annotated transcription start site.

  15. Applications of Recombinant Dna Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part B: Eukaryotic Gene Transcription and Post-Transcripional Rna Processing

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available The transcription of DNA into RNA is the primary level at which gene expression is controlled in eukaryotic cells. Eukaryotic gene transcription  involves several different RNA polymerases that interact with a host of transcription factors to initiate transcription. Genes that encode proteins are transcribed into messenger RNA (mRNA by RNA polymerase II. Ribosomal RNAs (rRNAs and transfer RNAs (tRNAs are transcribed by RNA polymerase I and III, respectively.  The production of each mRNA in human cells involves complex interactions of proteins (ie, trans-acting factors with specific sequences on the DNA (ie, cis-acting elements. Cis-acting elements are short base sequences adjacent to or within a particular gene. While the regulation of transcription is a pivotal step in the control of gene expression, a variety of molecular events, collectively known as ’RNA processing’  add an additional level of control of gene expression in eukaryotic cells.

  16. Automated Identification of Core Regulatory Genes in Human Gene Regulatory Networks.

    Directory of Open Access Journals (Sweden)

    Vipin Narang

    Full Text Available Human gene regulatory networks (GRN can be difficult to interpret due to a tangle of edges interconnecting thousands of genes. We constructed a general human GRN from extensive transcription factor and microRNA target data obtained from public databases. In a subnetwork of this GRN that is active during estrogen stimulation of MCF-7 breast cancer cells, we benchmarked automated algorithms for identifying core regulatory genes (transcription factors and microRNAs. Among these algorithms, we identified K-core decomposition, pagerank and betweenness centrality algorithms as the most effective for discovering core regulatory genes in the network evaluated based on previously known roles of these genes in MCF-7 biology as well as in their ability to explain the up or down expression status of up to 70% of the remaining genes. Finally, we validated the use of K-core algorithm for organizing the GRN in an easier to interpret layered hierarchy where more influential regulatory genes percolate towards the inner layers. The integrated human gene and miRNA network and software used in this study are provided as supplementary materials (S1 Data accompanying this manuscript.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Bürglin Thomas R

    2008-03-01

    Full Text Available Abstract 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 of 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 also found in many protists suggests that this

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

    Science.gov (United States)

    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.

  1. Construction and expression of the eukaryotic expressed plasmid of MIC3 gene from Toxoplasma gondii in IBRS-2 cells

    Institute of Scientific and Technical Information of China (English)

    Tao JIANG; Donglin ZHANG; Hao NIE; Baoan YAO; Junlong ZHAO

    2008-01-01

    The sequence encoding MIC3 was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the vector pMD18-T. The tar-get gene was subcloned into the eukaryotic vector pcDNA3.1 after the identification of pMD18-T-MIC3 by enzyme digesting, PCR amplification and sequencing. Then the target recombinant plasmids pcMIC3 were transfected into IBRS-2 cells, and the positive cells con-taining pcMIC3 plasmids were obtained under the selec-tion of G418. The expressed proteins from the positive cells were detected by SDS-PAGE, Western blot and ELISA. The results showed that the DNA sequence iden-tity was 99.9% between amplified MIC3 and that from GenBank. The molecular weight of the recombinant MIC3 protein with good immuno-activity was about 39.2 ku. These available data would lay the foundation for further studies on DNA vaccine against Toxoplasma gondii.

  2. Vernalization: a model for investigating epigenetics and eukaryotic gene regulation in plants.

    Science.gov (United States)

    Schmitz, Robert J; Amasino, Richard M

    2007-01-01

    The transition from vegetative to reproductive development is a highly regulated process that, in many plant species, is sensitive to environmental cues that provide seasonal information to initiate flowering during optimal times of the year. One environmental cue is the cold of winter. Winter annuals and biennials typically require prolonged exposure to the cold of winter to flower rapidly in the spring. This process by which flowering is promoted by cold exposure is known as vernalization. The winter-annual habit of Arabidopsis thaliana is established by the ability of FRIGIDA to promote high levels of expression of the potent floral repressor FLOWERING LOCUS C (FLC). In Arabidopsis, vernalization results in the silencing of FLC in a mitotically stable (i.e., epigenetic) manner that is maintained for the remainder of the plant life cycle. The repressed "off" state of FLC has features characteristic of facultative heterochromatin. Upon passing to the next generation, the "off" state of FLC is reset to the "on" state. The environmental induction and mitotic stability of vernalization-mediated FLC repression as well as the subsequent resetting in the next generation provides a system for studying several aspects of epigenetic control of gene expression.

  3. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  4. Automated alignment-based curation of gene models in filamentous fungi

    NARCIS (Netherlands)

    Burgt, van der A.; Severing, E.I.; Collemare, J.A.R.; Wit, de P.J.G.M.

    2014-01-01

    Background Automated gene-calling is still an error-prone process, particularly for the highly plastic genomes of fungal species. Improvement through quality control and manual curation of gene models is a time-consuming process that requires skilled biologists and is only marginally performed. The

  5. Comparative genomic analysis reveals a diverse repertoire of genes involved in prokaryote-eukaryote interactions within the Pseudovibrio genus.

    Directory of Open Access Journals (Sweden)

    Stefano eRomano

    2016-03-01

    Full Text Available 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

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

    Science.gov (United States)

    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.

  7. Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing

    Directory of Open Access Journals (Sweden)

    Koepke Tyson

    2012-01-01

    yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. Conclusions This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB, genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

  8. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part A: Eukaryotic Gene Structure and DNA Replication

    Directory of Open Access Journals (Sweden)

    Gary E Wild

    2000-01-01

    Full Text Available Progress in the basic sciences of cell and molecular biology has provided an exciting dimension that has translated into clinically relevant information in every medical subspecialty. Importantly, the application of recombinant DNA technology has played a major role in unravelling the intricacies related to the molecular pathophysiology of disease. This series of review articles constitutes a framework for the integration of the database of new information into the core knowledge base of concepts related to the pathogenesis of gastrointestinal disorders and liver disease. The goal of this series of three articles is to review the basic principles of eukaryotic gene expression. The first article examines the role of DNA in directing the flow of genetic information in eukaryotic cells.

  9. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    Xichun Han; Hong Zhang; Mingku Jia; Gang Han; Weidong Jiang

    2004-01-01

    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.

  10. Expression of TIMP-3 Gene by Construction of a Eukaryotic Cell Expression Vector and Its Role in Reduction of Metastasis in a Human Breast Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    XichunHan; HongZhang; MingkuJia; GangHan; WeidongJiang

    2004-01-01

    The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453. Cellular & Molecular Immunology.

  11. Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph.

    Science.gov (United States)

    Zauner, S; Fraunholz, M; Wastl, J; Penny, S; Beaton, M; Cavalier-Smith, T; Maier, U G; Douglas, S

    2000-01-04

    Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these "cells within cells" lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)(7)AAG(6)A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

  12. A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution

    Directory of Open Access Journals (Sweden)

    Logsdon John M

    2007-02-01

    Full Text Available Abstract Background Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST corresponding to 853 unique clones, 5275 genome survey sequences (GSS, and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus. Results The analyses revealed a compact genome with few, if any, introns and very short 3' untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes – mostly encoding metabolic proteins – that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT. Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. Conclusion Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote

  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.

    Science.gov (United States)

    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.

  14. An extended phylogenetic analysis reveals ancient origin of "non-green" phosphoribulokinase genes from two lineages of "green" secondary photosynthetic eukaryotes: Euglenophyta and Chlorarachniophyta

    Directory of Open Access Journals (Sweden)

    Sekimoto Hiroyuki

    2011-09-01

    Full Text Available Abstract Background Euglenophyta and Chlorarachniophyta are groups of photosynthetic eukaryotes harboring secondary plastids of distinct green algal origins. Although previous phylogenetic analyses of genes encoding Calvin cycle enzymes demonstrated the presence of genes apparently not derived from green algal endosymbionts in the nuclear genomes of Euglena gracilis (Euglenophyta and Bigelowiella natans (Chlorarachniophyta, the origins of these "non-green" genes in "green" secondary phototrophs were unclear due to the limited taxon sampling. Results Here, we sequenced five new phosphoribulokinase (PRK genes (from one euglenophyte, two chlorarachniophytes, and two glaucophytes and performed an extended phylogenetic analysis of the genes based on a phylum-wide taxon sampling from various photosynthetic eukaryotes. Our phylogenetic analyses demonstrated that the PRK sequences form two genera of Euglenophyta formed a robust monophyletic group within a large clade including stramenopiles, haptophytes and a cryptophyte, and three genera of Chlorarachniophyta were placed within the red algal clade. These "non-green" affiliations were supported by the taxon-specific insertion/deletion sequences in the PRK alignment, especially between euglenophytes and stramenopiles. In addition, phylogenetic analysis of another Calvin cycle enzyme, plastid-targeted sedoheptulose-bisphosphatase (SBP, showed that the SBP sequences from two genera of Chlorarachniophyta were positioned within a red algal clade. Conclusions Our results suggest that PRK genes may have been transferred from a "stramenopile" ancestor to Euglenophyta and from a "red algal" ancestor to Chlorarachniophyta before radiation of extant taxa of these two "green" secondary phototrophs. The presence of two of key Calvin cycle enzymes, PRK and SBP, of red algal origins in Chlorarachniophyta indicate that the contribution of "non-green" algae to the plastid proteome in the "green" secondary phototrophs is

  15. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

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

    Directory of Open Access Journals (Sweden)

    Lina Li

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  18. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  19. Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

    Science.gov (United States)

    Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

    2012-07-01

    The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions

  20. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization......, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user....

  1. Gene Expression Measurement Module (GEMM) - A Fully Automated, Miniaturized Instrument for Measuring Gene Expression in Space

    Science.gov (United States)

    Pohorille, Andrew; Peyvan, Kia; Karouia, Fathi; Ricco, Antonio

    2012-01-01

    The capability to measure gene expression on board spacecraft opens the door to a large number of high-value experiments on the influence of the space environment on biological systems. For example, measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, and determine the metabolic bases of microbial pathogenicity and drug resistance. These and other applications hold significant potential for discoveries in space biology, biotechnology, and medicine. Supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measurement of expression of several hundreds of microbial genes from multiple samples. The instrument will be capable of (1) lysing cell walls of bacteria sampled from cultures grown in space, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA on a microarray and (4) providing readout of the microarray signal, all in a single microfluidics cartridge. The device is suitable for deployment on nanosatellite platforms developed by NASA Ames' Small Spacecraft Division. To meet space and other technical constraints imposed by these platforms, a number of technical innovations are being implemented. The integration and end-to-end technological and biological validation of the instrument are carried out using as a model the photosynthetic bacterium Synechococcus elongatus, known for its remarkable metabolic diversity and resilience to adverse conditions. Each step in the measurement process-lysis, nucleic acid extraction, purification, and hybridization to an array-is assessed through comparison of the results obtained using the instrument with

  2. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    Science.gov (United States)

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  3. Expression of acyl-lipid Delta12-desaturase gene in prokaryotic and eukaryotic cells and its effect on cold stress tolerance of potato.

    Science.gov (United States)

    Amiri, Reza Maali; Yur'eva, Natalia O; Shimshilashvili, Khristina R; Goldenkova-Pavlova, Irina V; Pchelkin, Vasiliy P; Kuznitsova, Elmira I; Tsydendambaev, Vladimir D; Trunova, Tamara I; Los, Dmitry A; Jouzani, Gholamreza Salehi; Nosov, Alexander M

    2010-03-01

    We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

  4. An investigation into eukaryotic pseudouridine synthases.

    Science.gov (United States)

    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".

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

    Science.gov (United States)

    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. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    Directory of Open Access Journals (Sweden)

    Wu Bai-Lin

    2009-10-01

    Full Text Available Abstract Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other

  7. Eukaryotic diversity in historical soil samples

    NARCIS (Netherlands)

    Moon-van der Staay, S.Y.; Tzeneva, V.A.; Staay, van der G.W.M.; Vos, de W.M.; Smidt, H.; Hackstein, J.H.P.

    2006-01-01

    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

  8. The Choice between MapMan and Gene Ontology for Automated Gene Function Prediction in Plant Science.

    Science.gov (United States)

    Klie, Sebastian; Nikoloski, Zoran

    2012-01-01

    Since the introduction of the Gene Ontology (GO), the analysis of high-throughput data has become tightly coupled with the use of ontologies to establish associations between knowledge and data in an automated fashion. Ontologies provide a systematic description of knowledge by a controlled vocabulary of defined structure in which ontological concepts are connected by pre-defined relationships. In plant science, MapMan and GO offer two alternatives for ontology-driven analyses. Unlike GO, initially developed to characterize microbial systems, MapMan was specifically designed to cover plant-specific pathways and processes. While the dependencies between concepts in MapMan are modeled as a tree, in GO these are captured in a directed acyclic graph. Therefore, the difference in ontologies may cause discrepancies in data reduction, visualization, and hypothesis generation. Here provide the first systematic comparative analysis of GO and MapMan for the case of the model plant species Arabidopsis thaliana (Arabidopsis) with respect to their structural properties and difference in distributions of information content. In addition, we investigate the effect of the two ontologies on the specificity and sensitivity of automated gene function prediction via the coupling of co-expression networks and the guilt-by-association principle. Automated gene function prediction is particularly needed for the model plant Arabidopsis in which only half of genes have been functionally annotated based on sequence similarity to known genes. The results highlight the need for structured representation of species-specific biological knowledge, and warrants caution in the design principles employed in future ontologies.

  9. The choice between MapMan and Gene Ontology for automated gene function prediction in plant science

    Directory of Open Access Journals (Sweden)

    Sebastian eKlie

    2012-06-01

    Full Text Available Since the introduction of the Gene Ontology (GO, the analysis of high-throughput data has become tightly coupled with the use of ontologies to establish associations between knowledge and data in an automated fashion. Ontologies provide a systematic description of knowledge by a controlled vocabulary of defined structure in which ontological concepts are connected by pre-defined relationships. In plant science, MapMan and GO offer two alternatives for ontology-driven analyses. Unlike GO, initially developed to characterize microbial systems, MapMan was specifically designed to cover plant-specific pathways and processes. While the dependencies between concepts in MapMan are modeled as a tree, in GO these are captured in a directed acyclic graph. Therefore, the difference in ontologies may cause discrepancies in data reduction, visualization, and hypothesis generation. Here provide the first systematic comparative analysis of GO and MapMan for the case of the model plant species Arabidopsis thaliana (Arabidopsis with respect to their structural properties and difference in distributions of information content. In addition, we investigate the effect of the two ontologies on the specificity and sensitivity of automated gene function prediction via the coupling of coexpression networks and the guilt-by-association principle. Automated gene function prediction is particularly needed for the model plant Arabidopsis in which only half of genes have been functionally annotated based on sequence similarity to known genes. The results highlight the need for structured representation of species-specific biological knowledge, and warrants caution in the design principles employed in future ontologies.

  10. Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

    Science.gov (United States)

    Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

    2012-01-01

    Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of

  11. 乙型肝炎病毒X基因载体的构建及鉴定%Construction and identification of HBV X gene eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    付逢萍; 白桂芹; 刘妍

    2011-01-01

    目的 构建乙型肝炎病毒(HBV)X基因高效真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及乙型肝炎病毒母婴垂直传播的可能机制奠定基础.方法 设计并合成乙型肝炎病毒X基因的引物,以我国急性肝炎患者血清中获得的全长乙型肝炎病毒序列C基因型作为构建载体X基因序列的母板进行扩增,将扩增产物X基因连接到真核表达载体pcDNA3.1(+)上,酶切图谱分析、PCR检测和扩增产物序列分析等鉴定所构建的真核表达载体.结果 以重组载体为模板、用乙型肝炎病毒X基因引物PCR扩增得到的目的 片段为500bp,与已知乙型肝炎病毒X基因大小相同,酶切后发现在500bp左右及5kb左右出现两条特异性条带,分别与已知X基因序列相同.结论 成功构建了乙型肝炎病毒X基因真核表达载体,为进一步研究乙型肝炎病毒X基因的功能及宫内感染的可能机制奠定了基础.%Objective To construct a highly effective eukaryotic expression vector pcDNA3. 1( + )with HBV X gene[ pcDNA3. 1( + )-X ],and then to study the function of HBV X gene and the probable mechanism of intrauterine infection. Methods Primers of HBV X gene were designed. All HBV sequences genotype C as mother blank. HBV X gene was amplified by PCR. The PCR products were connected to the eukaryotic expression vectors pCDNA3. 1( + ) by restric:tion endonucleases and T4 DNA joining enzyme. Restriction endonucleases digest analysis, PCR analysis , and sequence analysis were used to identify the recombinants. Results Taking reconstructed vector as mould, the fragment amplified by PCR was 500bp, which was same as known HBV X gene. After treated with restriction endonucleases, two specitic bands presented around 500bp and 5kb, which were same as know X gene sequence. Conclusion The eukaryotic expression vector pCDNA3. 1( + )-X has been successfully constructed, which provides some foundation for analysis of the function of HBV X

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

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    Yiling Liu

    2015-11-01

    Full Text Available 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.

  14. Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang

    2005-01-01

    Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

  15. Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

    Science.gov (United States)

    Liu, Robin H.; Lodes, Mike; Fuji, H. Sho; Danley, David; McShea, Andrew

    Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed

  16. TOGA: An automated parsing technology for analyzing expression of nearly all genes

    OpenAIRE

    Sutcliffe, J. Gregor; Foye, Pamela E.; Erlander, Mark G.; HIlbush, Brian S.; Bodzin, Leon J.; Durham, Jayson T.; Hasel, Karl W.

    2000-01-01

    We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3′ ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tis...

  17. Depth matters: Microbial eukaryote diversity and community structure in the eastern North Pacific revealed through environmental gene libraries

    Science.gov (United States)

    Schnetzer, Astrid; Moorthi, Stefanie D.; Countway, Peter D.; Gast, Rebecca J.; Gilg, Ilana C.; Caron, David A.

    2011-01-01

    Protistan community structure was examined from 6 depths (1.5, 20, 42, 150, 500, 880 m) at a coastal ocean site in the San Pedro Channel, California. A total of 856 partial length 18S rDNA protistan sequences from the six clone libraries were analyzed to characterize diversity present at each depth. The sequences were grouped into a total of 259 Operational Taxonomic Units (OTUs) that were inferred using an automated OTU calling program that formed OTUs with approximately species-level distinction (95% sequence similarity). Most OTUs (194 out of 259) were observed at only one specific depth, and only two were present in clone libraries from all depths. OTUs were obtained from 21 major protistan taxonomic groups determined by their closest BLAST matches to identified protists in the NCBI database. Approximately 74% of the detected OTUs belonged to the Chromalveolates, with Group II alveolates making up the largest single group. Protistan assemblages at euphotic depths (1.5, 20 and 42 m) were characterized by the presence of clades that contained phototrophic species (stramenopiles, chlorophytes and haptophytes) as well as consumers (especially ciliates). Assemblages in the lower water column (150, 500 and 800 m) were distinct from communities at shallow depths because of strong contributions from taxa belonging to euglenozoans, acantharians, polycystines and Taxopodida ( Sticholonche spp. and close relatives). Species richness (Chao I estimate) and diversity (Shannon index) were highest within the euphotic zone and at 150 m, and lowest for protistan assemblages located in the oxygen minimum zone (500 and 880 m). Multivariate analyses (Bray-Curtis coefficient) confirmed that protistan assemblage composition differed significantly when samples were grouped into shallow (≤150 m) and deep water assemblages (≥150 m).

  18. In vitro Expression in Eukaryotic Cells of a Prion Protein Gene Cloned from Scrapie-Infected Mouse Brain

    Science.gov (United States)

    Caughey, Byron; Race, Richard E.; Vogel, Mari; Buchmeier, Michael J.; Chesebro, Bruce

    1988-07-01

    It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

  19. GSMA: Gene Set Matrix Analysis, An Automated Method for Rapid Hypothesis Testing of Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Chris Cheadle

    2007-01-01

    Full Text Available Background: Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The assignment of functional information to these complex patterns remains a challenging task in effectively interpreting data and correlating results from across experiments, projects and laboratories. Methods which allow the rapid and robust evaluation of multiple functional hypotheses increase the power of individual researchers to data mine gene expression data more efficiently.Results: We have developed (gene set matrix analysis GSMA as a useful method for the rapid testing of group-wise up- or downregulation of gene expression simultaneously for multiple lists of genes (gene sets against entire distributions of gene expression changes (datasets for single or multiple experiments. The utility of GSMA lies in its flexibility to rapidly poll gene sets related by known biological function or as designated solely by the end-user against large numbers of datasets simultaneously.Conclusions: GSMA provides a simple and straightforward method for hypothesis testing in which genes are tested by groups across multiple datasets for patterns of expression enrichment.

  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.

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  2. Bacterial proteins pinpoint a single eukaryotic root.

    Science.gov (United States)

    Derelle, Romain; Torruella, Guifré; Klimeš, Vladimír; Brinkmann, Henner; Kim, Eunsoo; Vlček, Čestmír; Lang, B Franz; Eliáš, Marek

    2015-02-17

    The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively.

  3. 人CCR5基因真核表达质粒的构建及其鉴定%Construction and characterization of plasmid expressing human CCR5 gene in eukaryotes

    Institute of Scientific and Technical Information of China (English)

    程林; 宋红勇; 吴喜林; 吴稚伟

    2012-01-01

    目的:构建人CCR5基因的真核表达质粒并对其进行功能鉴定.方法:PCR扩增人CCR5基因,将其克隆入真核表达载体pcDNA3.1内,构建含人CCR5基因的真核表达质粒pcDNA3.1-CCR5.使用RT-PCR、流式细胞术和HIV假病毒感染实验的方法,鉴定CCR5在真核细胞中的表达和功能.结果:克隆的人CCR5基因与GenBank中已登记的基因序列100%同源.瞬时转染真核细胞后,RT-PCR在预期的位置检测出目的条带,流式细胞术检测到约25.6%的细胞表达CCR5蛋白,且该蛋白能介导HIV假病毒的感染.结论:成功构建了含人CCR5基因的真核表达质粒.%Objective:To construct and characterize a eukaryotic system for expressing human CCR5 gene. Methods; Human CCR5 gene was amplified by PCR, and subcloned into pcDNA3.1 vector to construct a recombinant plasmid pcDNA3. 1-CCR5. The expression of human CCR5 gene in eukaryotic cells was verified by RT-PCR and flow cytometry. HIV-1 env pseudotyped virus infection assay was used to detect the function of CCR5 gene in eukaryotic cells. Results:The sequence of inserted CCR5 gene fragment was 100% homology compared to human CCR5 gene registered in GenBank. After transfection of eukaryotic cells with pcDNA3. 1-CCR5, the target band was identified by RT-PCR and about 25. 6% of the CCR5 protein was detected by flow cytometry. Furthermore, the protein could mediate HIV pseudotype virus infection. Conclusion:A functional eukaryotic expression plasmid pcDNA3. 1-CCR5 has been established successfully.

  4. Autophagy in unicellular eukaryotes

    NARCIS (Netherlands)

    Kiel, J.A.K.W.

    2010-01-01

    Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components

  5. The Genome of Naegleria gruberi Illuminates Early Eukaryotic Versatility

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Eukaryotic diversity in historical soil samples.

    Science.gov (United States)

    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.

  7. Automated Extraction Of Associations Between Methylated Genes and Diseases From Biomedical Literature

    KAUST Repository

    Bin Res, Arwa A.

    2012-12-01

    Associations between methylated genes and diseases have been investigated in several studies, and it is critical to have such information available for better understanding of diseases and clinical decisions. However, such information is scattered in a large number of electronic publications and it is difficult to manually search for it. Therefore, the goal of the project is to develop a machine learning model that can efficiently extract such information. Twelve machine learning algorithms were applied and compared in application to this problem based on three approaches that involve: document-term frequency matrices, position weight matrices, and a hybrid approach that uses the combination of the previous two. The best results we obtained by the hybrid approach with a random forest model that, in a 10-fold cross-validation, achieved F-score and accuracy of nearly 85% and 84%, respectively. On a completely separate testing set, F-score and accuracy of 89% and 88%, respectively, were obtained. Based on this model, we developed a tool that automates extraction of associations between methylated genes and diseases from electronic text. Our study contributed an efficient method for extracting specific types of associations from free text and the methodology developed here can be extended to other similar association extraction problems.

  8. Comparative genomics and evolution of eukaryotic phospholipidbiosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Transfer of DNA from Bacteria to Eukaryotes

    Directory of Open Access Journals (Sweden)

    Benoît Lacroix

    2016-07-01

    Full Text Available 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.

  10. TOGA: an automated parsing technology for analyzing expression of nearly all genes.

    Science.gov (United States)

    Sutcliffe, J G; Foye, P E; Erlander, M G; Hilbush, B S; Bodzin, L J; Durham, J T; Hasel, K W

    2000-02-29

    We have developed an automated, high-throughput, systematic cDNA display method called TOGA, an acronym for total gene expression analysis. TOGA utilizes 8-nt sequences, comprised of a 4-nt restriction endonuclease cleavage site and adjacent 4-nt parsing sequences, and their distances from the 3' ends of mRNA molecules to give each mRNA species in an organism a single identity. The parsing sequences are used as parts of primer-binding sites in 256 PCR-based assays performed robotically on tissue extracts to determine simultaneously the presence and relative concentration of nearly every mRNA in the extracts, regardless of whether the mRNA has been discovered previously. Visualization of the electrophoretically separated fluorescent assay products from different extracts displayed via a Netscape browser-based graphical user interface allows the status of each mRNA to be compared among samples and its identity to be matched with sequences of known mRNAs compiled in databases.

  11. Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9.

    Science.gov (United States)

    Attayek, Peter J; Waugh, Jennifer P; Hunsucker, Sally A; Grayeski, Philip J; Sims, Christopher E; Armistead, Paul M; Allbritton, Nancy L

    2017-05-15

    Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.

  12. BMP2和TGFβ3双基因真核表达载体的构建%Construction of bicistronic eukaryotic expression plasmid containing BMP2 and TGFβ3 genes

    Institute of Scientific and Technical Information of China (English)

    马小松; 刘金钊; 王昌耀; 王英振; 刘世海; 刘相萍

    2011-01-01

    [目的]构建骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体.[方法]从人胚胎组织提取总RNA,反转录成cDNA,以pGEMT/BMP2和反转录的cDNA为模板,PCR扩增出BMP2和TGFβ3两个基因全长,将两个基因片段分别定向连入双基因真核表达载体pIRES;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.[结果]酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.[结论]成功构建pIRESBMP2/TGFβ3双基因真核表达载体.%[ Objective]To construct a bicistronic eukayotic expression plasmid consisting of BMP2 and TGFβ3 target genes.[ Method ] The DNA fragment of BMP2 and TGFβ3 genes were obtained from pGEMT/BMP2 plasmid and human embryonal tissue by PCR, respectively. They were inserted into bicistronic eukaryotic expression plasmid vector plRES. The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing. [ Result ] The direction and sequences of the new bicistronic eukaryotic expression plasmid pIRES - BMP2 -TGFβ3 were correct. [ Conclusion] The bicistronic eukaryotic expression plasmid has been constructed successfully.

  13. Eukaryotic membrane protein overproduction in Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Chan, Ka Wai; Slotboom, Dirk Jan; Floyd, Suzanne; O’Connor, Rosemary; Monné, Magnus

    2005-01-01

    Eukaryotic membrane proteins play many vital roles in the cell and are important drug targets. Approximately 25% of all genes identified in the genome are known to encode membrane proteins, but the vast majority have no assigned function. Although the generation of structures of soluble proteins has

  14. 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.

    Directory of Open Access Journals (Sweden)

    Hisayoshi Nozaki

    Full Text Available 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.

  15. Atypical mitochondrial inheritance patterns in eukaryotes.

    Science.gov (United States)

    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.

  16. Eukaryotic association module in phage WO genomes from Wolbachia

    Science.gov (United States)

    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

  17. The COG database: an updated version includes eukaryotes

    Directory of Open Access Journals (Sweden)

    Sverdlov Alexander V

    2003-09-01

    Full Text Available Abstract Background The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. Results We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens, one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the

  18. Statistical characteristics of eukaryotic intron database

    Institute of Scientific and Technical Information of China (English)

    HE Miao; LI Jidong; ZHANG Shanghong

    2006-01-01

    A database called eukaryotic intron database (EID) was developed based on the data from GenBank.Studies on the statistical characteristics of EID show that there were 103,848 genes,478,484 introns,and 582,332 exons,with an average of 4.61 introns and 5.61 exons per gene.Introns of 40-120 nt in length were abundant in the database.Results of the statistical analysis on the data from nine model species showed that in eukaryotes,higher species do not necessarily have more introns or exons in a gene than lower species.Furthermore,characteristics of EID,such as intron phase,distribution of different splice sites,and the relationship between genome size and intron proportion or intron density,have been studied.

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

    Energy Technology Data Exchange (ETDEWEB)

    Koonin, Eugene V., E-mail: koonin@ncbi.nlm.nih.gov [National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 (United States); Dolja, Valerian V., E-mail: doljav@science.oregonstate.edu [Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 (United States); Krupovic, Mart, E-mail: krupovic@pasteur.fr [Institut Pasteur, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, Paris 75015 (France)

    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

  20. 肝片吸虫GST真核表达载体构建及重组蛋白活性分析%Construction on eukaryotic expression plasmid of Fasciola hepatica GST gene and analysis on recombinant protein

    Institute of Scientific and Technical Information of China (English)

    冉旭华; 闻晓波; 王春仁; 刘娣; 孙中武; 李晓娟; 王密

    2011-01-01

    目的 构建肝片吸虫谷胱甘肽S-转移酶(GST)的真核表达载体,研究重组蛋白的免疫原性.方法 以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因(GST),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究.%In this research, we constructed eukaryotic expression plasmid of Fasciola hepatica GST gene and analyzed the immunogenicity of recombinant protein. The glutathione S-transferase (GST) gene of F. hepatica played the importanty protective role against the worms infection. In this research,GST was amplified by PCR from the pET30a-FhGST, and then inserted into pEGFP-Nl vector to construct recombinant plasmids pEGFP-GST. The recombinant plasmid pEGFP-GST was transfected into Hela cells and fluorescent signal was detected by fluorescence microscope. Western blotting analysis was done to analysze immunogenicity of recombinant protein. And the results demonstrated that eukaryotic expression plasmid of Fasciola hepatica GST gene was constructed successfully. Recombinant protein could be specifically recognized by goat serum infected by Fasciola hepatica, which proving its immunoreactivity. It's suggested that the eukaryotic expression plasmid might be used as gene vaccine in further research.

  1. Molecular Data are Transforming Hypotheses on the Origin and Diversification of Eukaryotes.

    Science.gov (United States)

    Tekle, Yonas I; Parfrey, Laura Wegener; Katz, Laura A

    2009-06-01

    The explosion of molecular data has transformed hypotheses on both the origin of eukaryotes and the structure of the eukaryotic tree of life. Early ideas about the evolution of eukaryotes arose through analyses of morphology by light microscopy and later electron microscopy. Though such studies have proven powerful at resolving more recent events, theories on origins and diversification of eukaryotic life have been substantially revised in light of analyses of molecular data including gene and, increasingly, whole genome sequences. By combining these approaches, progress has been made in elucidating both the origin and diversification of eukaryotes. Yet many aspects of the evolution of eukaryotic life remain to be illuminated.

  2. 犬SLAM基因真核表达载体的构建及Vero细胞系转染的研究%Construction of Eukaryotic Expressing Vector of SLAM Gene and its Transient Expression in Vero Cell

    Institute of Scientific and Technical Information of China (English)

    苏建青; 褚秀玲; 张吉清; 马秀亮; 江成

    2012-01-01

    To elucidate the character and functions of cellular receptors of canine distemper virus, the gene SLAM of canine distemper virus' s open reading frame (ORF) was cloned into eukaryotic expression vector pCDNA3.1(+) to generate the recombinant plasmid pcDNA3.1/S.LAM. The pureed plasmid was transected into Vero cells in vitro with Lipofectamine 2000. The transient expression of the SLAM protein was detected by reverse transcription polymerase chain reaction and Westem-blot assay. The results showed that the eukaryotic expression vectors of canine SLAM gene were constructed successfully. The reverse transcription polymerase chain reaction and Western-blot assay confirmed that the protein SLAM was expression in Vero cell. Recombinant SLAM was successfully expressed, which laid foundation for further research on the stable express of SLAM gene in Vero.%为了研究犬瘟热病毒细胞受体SLAM基因的特性和功能.将基因SLAM克隆到真核表达载体pcDNA3.1(+)上,构建真核表达载体pCDNA3.1/SLAM,重组质粒纯化后应用脂质体2000转染到Vero细胞中,通过RT-PCR和免疫印迹方法检测犬SLAM基因在Vero细胞中的转录和表达情况.结果表明:成功构建了表达SLAM基因的真核表达载体,RT-PCR和免疫印迹显示SLAM基因获得表达;pcDNA3.1/SLAM载体的构建为研究犬SLAM基因在Vero细胞中的稳定表达奠定了基础.

  3. Construction and Expression of Eukaryotic Expression vector containing Hantaan Virus S gene and CpG motif%插入CpG序列的汉坦病毒S基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    郑兰艳; 罗恩杰; 李新鸣; 牟玲; 张小棠; 鲁润铭

    2004-01-01

    To improve the effect of the gene immunization against Hantaan virus, we constructed the eukaryotic expression vector pTARGET-hans(ISS) containing Hantaan Virus S gene coding region and CpG motif by cloning S gene segment with CpG motif into eukaryotic expression vector pTARGETTM.After conformed by enzyme analysis, the recombinant expression vector pTARGET-hans(ISS) was transferred into Vero-E6 cells by electroporation and the transient expression of Hantaan virus nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transferred Vero-E6, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector pTARGET-hans(ISS) was successfully constructed and expressed in vitro,which will lay a foundation for further animal vaccination.

  4. 沙眼衣原体ompA基因克隆及其在真核细胞中的表达%Cloning of ompA gene from Chlamydia trachomatis and its expression in eukaryotic cell

    Institute of Scientific and Technical Information of China (English)

    李忠玉; 吴移谋; 陈超群; 余敏君

    2004-01-01

    目的克隆D型沙眼衣原体(Ct)ompA基因,构建真核表达重组质粒,转染真核细胞,为核酸疫苗的研制作准备.方法用PCR技术从D型Ct基因组DNA中扩增ompA基因片段,重组入pUCm-T克隆载体.将pUCm-T/ompA中的ompA外源基因片段经酶切、连接等反应,亚克隆入pcDNA3.1真核表达载体,进行序列分析和酶切鉴定后,运用脂质体将重组体pcDNA3.1/ompA转染HeLa细胞,免疫组化法观察目的的基因的表达.结果从D型Ct基因组DNA中扩增出特异的ompA基因片段;序列测定证实与GenBank登陆的D型Ct一致;重组质粒pcDNA3.1/ompA在HeLa中获得表达.结论Ct ompA基因能够在体外真核细胞表达,为进一步研究Ct致病机制及DNA疫苗的研究提供理论依据.%Objective: To clone and construct the recombinant plasmid containing ompA gene from Chlamydia trachomatis, and transfect it into mammalian cells to express the major outer membrane protein (MOMP). Methods: Amplifed from the genomic DNA of Chlamydia trachomatis using polymerase chain reaction (PCR), ompA gene was inserted into cloning vector pUCm-T. The inserted ompA gene was subcloned to pcDNA3.1 eukaryotic expression vector by linking reactions. After identifying by sequencing and restrictive enzymes digestion. The recombinant plasmid was transfected into HeLa cells using Liposome. Results: The specific gene fragment about 1.2 kb was successfully amplified. The DNA sequence of ompA gene was found to be the same as the nucleotide sequence published by GenBank. Immunocytochemistry analysis showed that om pA gene was expressed in HeLa cells and located in cytoplasm. Conclusion: OmpA gene can be expressed in eukaryotic system which lay the foundation for studing the pathogenic mechanism and the development of the Chlamydia trachomatis vaccine against this pathogen.

  5. Construction of eukaryotic expression vecter for human ski gene and observation of its role in promoting cell proliferation%人ski基因真核表达载体的构建及促细胞增殖作用

    Institute of Scientific and Technical Information of China (English)

    彭艳; 李平; 刘苹; 陈磊; 赵晓光; 周元国

    2011-01-01

    背景:c-Ski 既可促进组织修复,又能降低瘢痕形成,有望成为一个全新的基因治疗药物.目的:构建人ski 基因的真核表达载体,并对其促成纤维细胞增殖效果进行验证.方法:RT-PCR 法从人包皮培养原代成纤维细胞总RNA 中扩增出人ski 基因,连同真核表达启动子CMV 克隆到pUC118 载体上,酶切和测序验证后,应用脂质体将其转染到培养的大鼠皮肤成纤维细胞中.结果与结论:酶切和测序结果证实表达质粒构建成功,Western blot 显示Ski 蛋白在转染细胞中成功表达,且可显著提高转染细胞的增殖效应,说明以pUC118 为骨架的重组ski 表达质粒具有促进大鼠皮肤成纤维细胞增殖的作用.%BACKGROUND: c-Ski can promote tissue repair and alleviate scar formation, which is expected to be a new gene therapeutic drugs. OBJECTIVE: To construct eukaryotic expression plasmid of human ski gene and to investigate its proliferative role in fibroblasts. METHODS: The ski gene was amplified by polymerase chain reaction (PCR) from human foreskin cultured fibroblast and was sub-cloned into pUC118 with CMV eukaryotic promoter. The recombinant pUC118-Ski plasmids were transfected into rat skin fibroblasts using lipofectamine TM 2000 and then its protein expression and effect of the proliferation were detected. RESULTS AND CONCLUSION: pUC118-Ski vector was constructed successfully which was confirmed by restriction endonuclease digestion and DNA sequencing. It expressed successfully and significantly improved the effect of the proliferation in transfected fibroblasts. Recombinant eukaryotic expression plasmid pUC118-Ski can improve the proliferation of rat skin fibroblasts.

  6. 人IL-37b基因真核表达载体的构建与表达%Construction and expression of eukaryotic expression vector of human IL-37 b gene

    Institute of Scientific and Technical Information of China (English)

    姚静; 程江; 裴雪枫; 王静宇; 袁明

    2016-01-01

    目的:构建pEGFP-N1/IL-37b真核表达载体,并检测其在THP-1细胞中的表达情况。方法从人PBMCs中提取总RNA,利用RT-qPCR技术扩增出IL-37b基因编码区序列,克隆至pEGFP-N1真核表达载体,将构建的重组质粒pEGFP-N1/IL-37b转染到THP-1细胞中,通过RT-qPCR和Western blot检测IL-37的表达。结果双酶切及基因测序结果显示IL-37b基因正确插入真核表达载体pEGFP-N1中;RT-qPCR和Western blot结果显示转染THP-1细胞后,IL-37表达水平明显升高(P<0.01)。结论成功构建了新型抗炎因子IL-37真核表达载体pEG-FP-N1/IL-37b,为进一步研究IL-37功能及与相关疾病的关系奠定基础。%Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells ( PB-MCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eu-karyotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detec-ted by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1. RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P<0. 01) after transfection in THP-1 cells. Conclusions We successful-ly constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foun-dation for further study on IL-37 functions and its association with related diseases.

  7. GeNeDA: An Open-Source Workflow for Design Automation of Gene Regulatory Networks Inspired from Microelectronics.

    Science.gov (United States)

    Madec, Morgan; Pecheux, François; Gendrault, Yves; Rosati, Elise; Lallement, Christophe; Haiech, Jacques

    2016-10-01

    The topic of this article is the development of an open-source automated design framework for synthetic biology, specifically for the design of artificial gene regulatory networks based on a digital approach. In opposition to other tools, GeNeDA is an open-source online software based on existing tools used in microelectronics that have proven their efficiency over the last 30 years. The complete framework is composed of a computation core directly adapted from an Electronic Design Automation tool, input and output interfaces, a library of elementary parts that can be achieved with gene regulatory networks, and an interface with an electrical circuit simulator. Each of these modules is an extension of microelectronics tools and concepts: ODIN II, ABC, the Verilog language, SPICE simulator, and SystemC-AMS. GeNeDA is first validated on a benchmark of several combinatorial circuits. The results highlight the importance of the part library. Then, this framework is used for the design of a sequential circuit including a biological state machine.

  8. α-葡萄糖苷酶基因序列分析及真核表达载体构建%Sequence Analysis of the Gene Encoding α-glucosidase and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    王继瑞; 张云开; 覃晓娟; 李玮; 梁智群

    2009-01-01

    Objective:Sequence analysis of the gene encoding α-glucosidase(agdA) from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector.Method:A pair of specific primers were designed and synthesized.Using the genome DNA as template,the DNA fragment(D1)was amplified with PCR,and using the total RNA as template,the DNA fragment(D2)was amplified with RT-PCR.The two DNA fragment was cloned into Escherichia coli DH5α and sequenced.Sequence analysis of the gene was on BLAST.The cDNA of agdA was inserted to eukaryotic expression vector pGAPZαA to construct the recombinant α-glucosidase expression vector.Result:The agdA was 3 127bp,composed of three exons and four introns.The cDNA of agdA was 2 958bp,containing the complete coding frame,encoding 985 amino acids.The sequence of agdA was compared with the sequence of template gene,of which the location of 6 bases had changed.The result showed that the homology could reach 99%.The eukaryotic expression vector pGAPZαA-agdA had been constructed.Conclusion: Sequence analysis of the gene agdA from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector had layed a foundation for expressing α-glucosidase of Aspergillus niger(CU-1) in Pichia pastoris.%目的:Aspergillus niger(CU-1)菌株α-葡萄糖苷酶基因(aNA)克隆并对其序列进行分析,构建该基因的真核表达载体.方法:设计合成的一对特异性引物,采用PCR以总DNA为模板,扩增得到DNA片段(01);采用RT-PCR方法扩增得到DNA片段(D2).将DNA片段D1和D2转入大肠杆菌中并进行了序列测定,序列进行BLAST比对分析.将α-葡萄糖苷酶基因的cDNA片段与表达载体pGAeZαA连接,构建重组表达载体.结果:基因agdA大小为3 127bp,含有3个外显子和4个内含子.该基因的cDNA序列大小为2 958bp,包含完整的编码框,编码985个氨基酸.agdA基因序列与已发表的α-葡萄糖苷基因序列同源性达99%,其中有6个位置的碱基发生了变化.已成功

  9. Energetics and genetics across the prokaryote-eukaryote divide

    Directory of Open Access Journals (Sweden)

    Lane Nick

    2011-06-01

    Full Text Available Abstract 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

  10. 犬SLAM基因真核表达载体的构建及在MDCK细胞中的稳定表达%Construction of Eukaryotic Expressing Vector of SLAM Gene and Establishment of Its Stable Expressing MDCK Cell

    Institute of Scientific and Technical Information of China (English)

    褚秀玲; 苏建青; 江成; 张吉清; 马秀亮

    2012-01-01

    To construction a MDCK cell line stably expressing signalling lymphocyte activation molecules (SLAM). The SLAM gene of cellular receptor of CDV was amplified by RT-PCR from canine peripheral blood lymphocytes. The correctly identified SLAM gene was inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/ SLAM. The pcDNA3.1/SLAM was transfected into MDCK cells by Lipofectamine. The stably expressing MDCK cell was screened with DMEM medium under the drug selection of G418. The single clone strain was purified by limiting dilution. The results indicated that the eukaryotic expression vector pcDNA3,l/SLAM was successfully constructed, then the stable expressing MDCK cell line was obtained.%为了构建稳定表达犬信号淋巴细胞激活因子(SLAM)的MDCK细胞系,该研究从犬外周血淋巴细胞中克隆了犬瘟热病毒(Canine distemper virus,CDV)细胞受体SLAM基因,将鉴定正确的SLAM基因插入到高效真核表达载体pcDNA3.1(+)中.采用脂质体转染的方式将重组质粒pcDNA3.1/SLAM转染到MDCK细胞中,采用G418加压筛选及有限稀释法克隆,获取稳定表达SLAM的MDCK细胞株.结果表明,成功构建了SLAM的真核表达载体pcDNA3.1/SLAM,并通过G418筛选获得了稳定表达SLAM的细胞系MDCK.

  11. Construction and Identification of mouse alpha-enolase enzyme gene eukaryotic expression vector%小鼠α烯醇化酶基因真核表达载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    贾卫红; 罗德炎; 段跃强; 李志奎; 王希良

    2011-01-01

    Objective To construct the eukaryotic expression vector of mouse alpha-enolase enzyme (Eno1)gene. Methods Enol eDNA of mouse kidney cells was amplified with polymerase chain reaction and inserted into the eukaryotic expression vector of pCDNA3. 1 +. The recombinant plasmid was verified by DNA sequencing and used to transfect Chinese hamster ovary(CHO)cells. Results The amplified fragment was 1 304bp in length. The sequence of Eno1 gene was in accordance with that in Genbank. The Enol protein was detected in CHO cells. Conclusion We successfully constructed the pCDNA3.1 +-Enol eukaryotic expression vector which may pave a way for further studies in vaccine experiment.%目的构建小鼠α烯醇化酶(Enol)基因的真核表达载体。方法以健康小鼠肾脏组织细胞的cDNA为模板,采用聚合酶链式反应(PCR)扩增Enol基因编码区的全部序列,克隆至真核细胞表达载体pCDNA3.1+中,测序鉴定目的基因用阳离子脂质体转染的方法转染中国仓鼠卵巢CHO细胞。结果PCR扩增的特异性片段长度为1 304bp,以此构建重组质粒pCDNA3.1+-Enol,测序结果与Genbank中的鼠源Enol基因cDNA序列一致。转染中国仓鼠卵巢细胞后可检测到Enol蛋白的表达。结论构建的真核表达载体pCDNA3.1+-Enol可在真核细胞内正确表达,这为进一步的疫苗研究奠定了基础。

  12. Establishment of hepatitis B virus X gene eukaryotic expression vector%乙型肝炎病毒X基因真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    李晓斐

    2012-01-01

    Objective To establish hepatitis B virus(HBV)X gene eukaryotic expression vector and identify its expression.Methods Polymerase chain reaction(PCR)was used to amplify X gene sequence,and the pcDNA6(+) vector and X gene PCR product double enzyme digestion transformed into Escherichia coli DH5 alpha.The positive clones were screened,and the double enzyme digestion and DNA sequencing were used to establish HBV X gene eukaryotic expression vector pcDNA6(+)-HBx.By polyetherimide(PEI)method,the transformation into HepG2 cells was performed,and Western blot identification of target protein was carried out.Results After pcDNA6-HBx enzyme digestion,agarose gel eleetrophoresis found HBx gene fragment,and sequencing results showed containing the complete X gene fragment.Western blot results showed that the pcDNA6(+)-HBx in HepG2 cells expressed X protein.Conclusions The eukaryotic expression vector pcDNA6(+)-HBx is established successfully,and there is X protein expression in HepG2 cells.It provides convenience for the further study of HBV X gene with chronic hepatitis and hepatocellular earcinonla.%目的 构建含有乙型肝炎病毒(HBV)X基因的真核表达载体,并对其表达进行鉴定.方法 用聚合酶链反应(PCR)方法扩增X基因序列,对pcDNA6(+ )载体及X基因PCR产物双酶切并转化大肠杆菌DH5α,筛选阳性克隆,并对其进行双酶切和测序鉴定,构建HBV X基因真核表达载体pcDNA6(+ )-HBx.以多聚乙烯酰胺(PEI)法将其转染到HepG2细胞,Western Blot鉴定目的 蛋白.结果 pcDNA6-HBx酶切后,琼脂糖电泳可见到HBx基因片段,经序列测定其含有完整的X基因片段;Western Blot结果显示pcDNA6(+ )-HBx能在HepG2细胞中表达X蛋白.结论 成功构建了真核表达载体pcDNA6(+)-HBx,并能在HepG2细胞中表达X蛋白,为进一步研究HBV X基因与慢性乙型肝炎及肝癌的关系提供便利条件.

  13. Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A mammalian expression plasmid pcDNA3.1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3.1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BamH Ⅰ and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot.Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3.1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3.1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.

  14. SIRT7基因乙酰化活性缺失突变真核表达载体的构建%The construction of eukaryotic expression vectors for SIRT 7 gene with acetylation activity deletion

    Institute of Scientific and Technical Information of China (English)

    李小娜; 梁慧敏; 王天晓; 韩飞; 刘文斌; 郑立娟; 董严; 刘晋祎; 时小燕

    2015-01-01

    Objective To construct eukaryotic expression vectors for SIRT 7 gene with acetylation activity deletion induced by site directed mutagenesis .Methods SIRT7 gene was amplified by RT‐PCR from 293T cells and cloned into the eukaryotic vector pcDNA 3 .1/myc‐His (‐) A . Site directed mutagenesis method based on two‐step PCR was performed to construct dominant negative (DN) mutants H187Y . The point mutation was verified by DNA sequencing . The mammalian 293T cell was transfected with SIRT7 and the mutants . The expression of the fused proteins was determined by Western blot .Results SIRT7 gene was cloned and the eukaryotic vector pcDNA 3 .1/myc‐His‐SIRT7 was constructed . The mutants were obtained by two‐step PCR .DNA sequencing showed that CAC at 560-562 bps sites , which encoded the 187 amino acids , was changed to TAC . No mutation was found in other base pair sites of the recombinant plasmids . The fusion protein myc‐SIRT7 was detectable by Western blot in 293T cells transfected with the mutant vectors . Conclusion The eukaryotic expression vectors for SIRT 7 and its mutants H187Y were successfully constructed .%目的:构建SIRT7基因乙酰化活性缺失的定点突变真核表达载体。方法用RT‐PCR法从293T细胞中扩增SIRT7基因,并克隆到真核载体 pcDNA 31./myc‐His(-)A上。采用 Two‐step PCR定点突变技术,构建SIRT7基因的 H187Y突变质粒,经测序确证定点突变成功,再将SIRT7及突变载体转染293T细胞,Western blot检测融合蛋白表达。结果克隆出SIRT7基因,构建了真核载体pcDNA 31./myc‐His‐SIRT7,经 Two‐step PCR获得突变体pcDNA 31./myc‐His‐SIRT7H187Y。DNA测序结果表明,编码187位氨基酸的560~562位碱基由CAC突变为TAC ,其他碱基均无突变。SIRT7和 H187Y突变载体转染293T 细胞后,可检测出myc‐SIRT7融合蛋白表达。结论成功构建了SIRT7以及 H187Y突变的真核表达载体。

  15. Expanding the eukaryotic genetic code

    Science.gov (United States)

    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.

  16. Expanding the eukaryotic genetic code

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-02-28

    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. Eukaryotic diversity at pH extremes.

    Science.gov (United States)

    Amaral-Zettler, Linda A

    2012-01-01

    Extremely acidic (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.

  18. Eukaryotic diversity at pH extremes

    Directory of Open Access Journals (Sweden)

    Linda A. Amaral-Zettler

    2013-01-01

    Full Text Available 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 7 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 Analysis (SIMPER 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.

  19. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  20. Endosymbiosis and Eukaryotic Cell Evolution.

    Science.gov (United States)

    Archibald, John M

    2015-10-05

    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.

  1. Automation of gene assignments to metabolic pathways using high-throughput expression data

    Directory of Open Access Journals (Sweden)

    Yona Golan

    2005-08-01

    Full Text Available Abstract Background Accurate assignment of genes to pathways is essential in order to understand the functional role of genes and to map the existing pathways in a given genome. Existing algorithms predict pathways by extrapolating experimental data in one organism to other organisms for which this data is not available. However, current systems classify all genes that belong to a specific EC family to all the pathways that contain the corresponding enzymatic reaction, and thus introduce ambiguity. Results Here we describe an algorithm for assignment of genes to cellular pathways that addresses this problem by selectively assigning specific genes to pathways. Our algorithm uses the set of experimentally elucidated metabolic pathways from MetaCyc, together with statistical models of enzyme families and expression data to assign genes to enzyme families and pathways by optimizing correlated co-expression, while minimizing conflicts due to shared assignments among pathways. Our algorithm also identifies alternative ("backup" genes and addresses the multi-domain nature of proteins. We apply our model to assign genes to pathways in the Yeast genome and compare the results for genes that were assigned experimentally. Our assignments are consistent with the experimentally verified assignments and reflect characteristic properties of cellular pathways. Conclusion We present an algorithm for automatic assignment of genes to metabolic pathways. The algorithm utilizes expression data and reduces the ambiguity that characterizes assignments that are based only on EC numbers.

  2. PatentMatrix: an automated tool to survey patents related to large sets of genes or proteins

    Directory of Open Access Journals (Sweden)

    de Rinaldis Emanuele

    2007-09-01

    Full Text Available Abstract Background The number of patents associated with genes and proteins and the amount of information contained in each patent often present a real obstacle to the rapid evaluation of the novelty of findings associated to genes from an intellectual property (IP perspective. This assessment, normally carried out by expert patent professionals, can therefore become cumbersome and time consuming. Here we present PatentMatrix, a novel software tool for the automated analysis of patent sequence text entries. Methods and Results PatentMatrix is written in the Awk language and requires installation of the Derwent GENESEQ™ patent sequence database under the sequence retrieval system SRS. The software works by taking as input two files: i a list of genes or proteins with the associated GENESEQ™ patent sequence accession numbers ii a list of keywords describing the research context of interest (e.g. 'lung', 'cancer', 'therapeutics', 'diagnostics'. The GENESEQ™ database is interrogated through the SRS system and each patent entry of interest is screened for the occurrence of user-defined keywords. Moreover, the software extracts the basic information useful for a preliminary assessment of the IP coverage of each patent from the GENESEQ™ database. As output, two tab-delimited files are generated which provide the user with a detailed and an aggregated view of the results. An example is given where the IP position of five genes is evaluated in the context of 'development of antibodies for cancer treatment' Conclusion PatentMatrix allows a rapid survey of patents associated with genes or proteins in a particular area of interest as defined by keywords. It can be efficiently used to evaluate the IP-related novelty of scientific findings and to rank genes or proteins according to their IP position.

  3. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.

    2014-11-21

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  4. Cloning of Dense Granular (GRA 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids

    Directory of Open Access Journals (Sweden)

    Zahra Arab-Mazar

    2014-11-01

    Full Text Available Background: Serological assay based on dense granular (GRA proteins of Toxoplasma gondii (T. gondii is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production.Materials and Methods: Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites’ DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing.Results: Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white (recombinant and blue (non-recombinant colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures.Conclusion: The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Steven W Paugh

    2016-02-01

    Full Text Available 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.

  7. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells.

    Science.gov (United States)

    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.

  8. Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Wanyi Li

    2014-03-01

    Full Text Available 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 TCID50 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 for

  9. Construction and expression of attenuated salmonella typhimurium carrying human adiponectin gene eukaryotic expression vector%重组人脂联素基因在减毒沙门氏菌中的表达

    Institute of Scientific and Technical Information of China (English)

    周静; 周洲; 吴莹; 向廷秀; 姜政; 王丕龙

    2009-01-01

    目的:构建携带人脂联素(AdipoQ)基因真核表达载体并在减毒沙门氏菌中表达,为AdipoQ对非酒精性脂肪性肝病(NAFLD)的基因治疗提供依据.方法:从人脂肪组织中提取总RNA,通过实时荧光定量PCR(rRT-PCR)方法获得Adi-poQ基因并将其克隆到真核表达载体pEGFP-NI上,构建pEG-FP-N1-AdipoQ重组载体.重组质粒经鉴定后再电转入减毒沙门氏菌SL7207中表达.结果:克隆的人AdipoQ基因744 bp测序结果显示:1个碱基发生突变,654位:A→T,突变率为0.1%,氨基酸Glu→Asp.经SDS-PAGE,Western Blot检测融合蛋白Mr约为55×10~3(绿色荧光蛋白Mr约为27×10~3),能够被AdipoQ抗体识别.结论:成功构建携带AdipoQ基因真核表达载体的减毒沙门氏菌株,AdipoQ基因能够在减毒沙门氏菌中表达并与绿色荧光蛋白融合.为进一步研究其在NAFLD中的作用机制奠定了基础.%AIM: To construct and express an attenuated Salmonella typhimurium strain contraining human adiponectin gene eukaryotic expression vector, in order to lay the foundation for the genetic therapy of nonalcoholic fatty liver disease in future. METHODS: Total mRNA was extracted from human fat tissue and the adiponectin cDNA was obtained by RT-PCR, and then cloned into eukaryotic expression vector pEGFP-N1. The recombinant vector was identified, and transformed into attenuated Salmonella typhimurium strain SL7207, and the recombinant strain SL7207/pEGFP-NI-AdipoQ was screened by green fluorescent microscope and Western Blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 744 bp, and had been inserted into eukaryotic expression vector, but as compared with gene reported by GenBank, 0. 1% of the gene mutation and O. 4% of amino acid residues change, respectively: 654 bp: A→T, amino acids: Glu→Asp. SDS-PAGE demonstrated that pEGFP-N1-AdipoQ was expressed in SL7207 strain, and the relatived molecular mass of the GFP-AdipoQ fusion protein

  10. 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.

    Science.gov (United States)

    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.

  11. Construction and Identification of the E gene Eukaryotic Expression Vector for Porcine Epidemic Diarrhea Virus%猪流行性腹泻病毒E基因真核表达载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    付通超; 万红平; 程渝; 颜其贵

    2012-01-01

    (目的)构建猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)E基因真核表达载体pCI—E,为探讨E基因的功能奠定基础。(方法)根据GenBank上已发表的PEDVCV777株序列,利用Primer5.0设计1对两端含限制性核酸内切酶EcoRI和SalI酶切位点的特异引物,用反转录聚合酶链式反应(RT—PCR)扩增出完整的231bp的目的基因,将目的基因与pMD19-T载体连接转化到大肠杆菌DH5a中,用测序、PCR、双酶切鉴定阳性克隆体。然后将纯化后的E基因插入表达载体(pCI—neo)上,并经双酶切、测序鉴定。(结果)成功构建了PEDV的pCI—E重组真核表达载体。(结论)pCI—E重组真核表达载体的构建为进一步探讨PEDVE基因的功能和分子生物学特性及PED防御新途径提供依据。%This study aimed to construct an eukaryotic expression vector of Porcine epidemic diarrhea virus E gene. Based on the published nucleotide sequence of PEDV in Genbank, a pair of specific primers of PEDV was designed by the Primer 5.0 software. To identify and clone the recombinant plasmid, we designed two restriction enzyme sites EcoR I and Sal I in the up- per and down primer, respectively. Following the mixture of the primers, template and the mix reaction system, the complete E gene of PEDV was amplified by RT--PCR. The fragment of the purpose gene was 231bp in length. The purpose gene was connected with pMD19-T vector, named pMD-E, transformed into E. coli DH5a. The positive colonies were confirmed by re- striction enzyme digestion, PCR and sequencing. A recombinant plasmid, named pCI-E was constructed by inserting the E gene into the expression vector pCI-neo, verified by restriction enzyme digestion and sequencing. The recombinant eukaryotic ex- pression vector of the PEDV E gene has been constructed successfully, which provided an experimental basis for the further study of the E gene functions and the PED defense.

  12. Evolutionary origin of eukaryotic cells.

    Science.gov (United States)

    Kostianovsky, M

    2000-01-01

    This article reviews literature on the transition from rudimentary prokaryotic life to eukaryotes. An overview of the differences between these organisms and theories of eukaryogenesis are reviewed. Various methods of investigating the transformation from prokaryotes to eukaryotes are elaborated, including the fossil, the molecular and living records, and examples are given. Lastly, the recent molecular studies and the impact on phylogenetic classification for the tree of life, based on molecular evolution, are discussed.

  13. Cloning and Expression of Spider Dragline Silk Protein Gene in Escherichia coli and Eukaryotic Cells%蜘蛛拖牵丝蛋白基因的克隆与表达

    Institute of Scientific and Technical Information of China (English)

    刘丹梅; 王芬; 李文利

    2011-01-01

    The extreme tensile strength and toughness of spider silk make it a superior candidate for a wide range of medical and industrial applications. In this study, a 837 bp fragment which encode dragline silk gene (ASP) was cloned from the genome of spider (Araneus ventricosus) and subcloned into pGEX-6p-l prokaryotic expression vector and pGFP-N2 eukaryotic expression vector, named pASG and pASN respectively. By induction at 16 ℃ for 24 h, the fusion protein GST-ASP was successfully expressed and characterized by Westem blotting with anti-GST antibodies. The fusion proteins ASP-GFP was also successfully expressed in insect sf9 cells by the detection of bright green fluorescence. That indicated the ASP gene was corrected expressed in E. coli and eukaryotic cells, respectively. This study provides a beneficial attempt to exploiting the pathway of producting spider silk protein by genetic engineering.%蜘蛛丝具有极高的强度和韧度,工业和医学应用价值很高,但由于蜘蛛的不可驯养性使其应用受到限制.因此,本文尝试利用基因工程的方法获得蛛丝蛋白的表达.我们利用巢式PCR技术从大腹圆蛛Araneus ventricosus基因组中克隆了长度为837 bp的拖牵丝蛋白基因(ASP),并分别将其构建至原核表达载体pGEX-6p-1和真核表达载体pGFP-N2上,分别命名为pASG和pASN.pASG在大肠杆菌中16℃下24 h诱导表达后,经蛋白质印迹证明成功地表达了GST-ASP融合蛋白;pASN转染昆虫sf9细胞48 h后观察到了绿色荧光蛋白GFP的表达,表明ASP基因在大肠杆菌和真核细胞中分别得到了正确表达.本研究为利用基因工程的方法开发蛛丝蛋白的生产途径提供了有益的尝试.

  14. A eukaryotic-acquired gene by a biotrophic phytopathogen allows prolonged survival on the host by counteracting the shut-down of plant photosynthesis

    KAUST Repository

    Garavaglia, Betiana S.

    2010-01-28

    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 downregulation 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. 2010 Garavaglia et al.

  15. Giant viruses and the origin of modern eukaryotes.

    Science.gov (United States)

    Forterre, Patrick; Gaïa, Morgan

    2016-06-01

    Several authors have suggested that viruses from the NucleoCytoplasmic Large DNA Viruses group (NCLDV) have played an important role in the origin of modern eukaryotes. Notably, the viral eukaryogenesis theory posits that the nucleus originated from an ancient NCLDV-related virus. Focusing on the viral factory instead of the virion adds credit to this hypothesis, but also suggests alternative scenarios. Beside a role in the emergence of the nucleus, ancient NCLDV may have provided new genes and/or chromosomes to the proto-eukaryotic lineage. Phylogenetic analyses suggest that NCLDV informational proteins, related to those of Archaea and Eukarya, were either recruited by ancient NCLDV from proto-eukaryotes and/or transferred to proto-eukaryotes, in agreement with the antiquity of NCLDV and their possible role in eukaryogenesis.

  16. Construction of the Antisense Eukaryotic Vector for Proliferating Cell Nuclear Antigen Gene and Its Expression in Bladder Cancer EJ Cell Line

    Institute of Scientific and Technical Information of China (English)

    童强松; 曾甫清; 齐义鹏; 朱朝晖; 鲁功成

    2002-01-01

    Summary: To explore a novel strategy for antisense gene therapy of cancer, the coding sequence ofhuman proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryoticvector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with li-posome. The PCNA expression in transferred cells was dynamically detected by immunofluo-rescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayedby MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryoticvector was successfully constructed and named as pLAPSN. After transfection with it for 1-7days, PCNA protein and mRNA levels in cancer cells were blocked by 16. 74 % - 84.21% (P<0. 05) and 23.27 % - 86.15 % (P<0. 05) respectively. The proliferation activities of transferredcells were inhibited by 27.91% - 62.07 % (P<0. 01), with cloning formation abilities being de-creased by 50. 81% (P<0. 01). It was concluded that the in vitro proliferation activities of cancercells could be effectively inhibited by blocking PCNA expression with antisense technique, whichcould serve as an ideal strategy for gene therapy of bladder cancer.

  17. Scheduling of Automated Guided Vehicle and Flexible Jobshop using Jumping Genes Genetic Algorithm

    Directory of Open Access Journals (Sweden)

    P. Paul Pandian

    2012-01-01

    Full Text Available Problem statement: Now a day’s many researchers try Genetic algorithm based optimization to find near optimal solution for flexible job shop. It is a global search. In Our study in the GA, some changes are made to search locally and globally by adding jumping genes operation. A typical flexible job shop model is considered for this research study. For that layout, five different example problems are formulated for purpose of evaluation. The material flow time for different shop types, processing times of products, waiting times of products, sequences of products are created and given in tabular form. Approach: The one of best evolutionary approach i.e., genetic algorithm with jumping genes operation is applied in this study, to optimize AGV flow time and the performance measures of Flexible Job shop manufacturing system. The non dominated sorting approach is used. Genetic algorithm with jumping genes operator is used to evaluate the method. Results: The AGV flow sequence is found out. Using this flow sequence make span, flow time of products with AGV, completion of the products is minimized. The position of the shop types are calculated for all products. The effectiveness of the proposed method is proved by comparing with Hamed Fazlollahtabar method. Conclusion: It is found that jumping genes genetic algorithm delivered good solutions as like as other evolutionary algorithms. Jumping genes genetic algorithm may applied to Multi objective optimization techniques in future.

  18. Construction of eukaryotic expression vector of recombinant human platelet CD36 gene and protein expression%重组人血小板CD36基因真核表达载体的构建及蛋白表达

    Institute of Scientific and Technical Information of China (English)

    付丽辉; 汪德清; 张杰; 孙春昀; 陈麟凤; 冯倩; 罗圆圆; 张晓娟; 王可; 于洋

    2012-01-01

    Objective To construct eukaryotic expression vector of recombinant human platelet CD36 gene , and to purified the functional protein of extracellular amino acid residues 30 to 439 segments. Methods The total of RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region (Gly30 ~ Asn439) residues amplified by RT-PCR. The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E. Coli DH5ct- We screened positive recombinant pMD18-CD36 plasmid. After sequencing, the gene inserted into the transient eukaryotic expression vector pTE2,constructed the pTE2-s-CD36-10 his transient eukaryotic expression vector. Then the recombinant CD36 Gly30 ~ Asn439 expressed by HEK-293 cells and was purified with Ni2 + 2NTA chromatography. Results 1.4 kb cDNA was amplified by RT-PCR,sequence analysis of the results was exactly the same as NM_001001547.2 in Genebank. Plasmid transfected HEK-293 cells, SDS-PAGE confirmed that cells expressed the human CD36 antigen extracellular protein fragments. Conclusion The CD36 Gly30 ~ Asn439 can be highly expressed by human embryonic kidney cells (HEK293). The purified protein should be pave the way for future study.%目的 制备具有功能活性的重组人血小板表面CD36抗原的纯化表达蛋白胞外区30 ~439氨基酸残基段.方法 提取人肝细胞组织总RNA,经RT-PCR扩增编码人血小板CD36抗原胞外区(Gly30~Asn439)氨基酸残基cDNA,构建于原核表达载体pMD18并转化大肠杆菌DH5α,筛选获得阳性重组子pMD18-CD36,提取质粒.经序列测定后,将该基因插入到真核细胞瞬时表达载体pTE2上,构建成为pTE2-s-CD36-10 his真核瞬时表达载体.采用lipofectamine 2000 (invitrogen)转染法,将重组质粒转染HEK-293细胞,表达产物经Ni2+ 2NTA柱层析纯化.结果 RT-PCR扩增获得了1.4kb的片段.invitrogen测序,该序列分析结果与Genebank中的NM_001001547.2完

  19. Soil eukaryotic functional diversity, a metatranscriptomic approach.

    Science.gov (United States)

    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.

  20. Distinct expression, localization and function of two Rab7 proteins encoded by paralogous genes in a free-living model eukaryote.

    Science.gov (United States)

    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.

  1. Construction of eukaryotic expression vector system for expression of VP1 gene of encephalomyocarditis virus%脑心肌炎病毒VP1基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    凡静静; 冯若飞; 杨妍梅; 张海霞; 李向茸; 王丹; 谢晶莹; 马忠仁

    2013-01-01

    The objective of this study was to construct a eukaryotic expression vector harboring VP1 gene of encephalomyocarditis virus(EMCV) ,and to express the VP1 gene in CHO cells. VP1 gene segment of EMCV was amplified by RT-PCR,and then cloned into the pEGFP-Cl vector to construct eukaryotic expression vector pEGFP-C1-VP1. The constructed plasmid was then transfected into the CHO cell via Lipo-fectamineTM 2000 Reagent. Transcription of VP1 gene in the CHO cell was tested via RT-PCR,and localization and reactinogenicity of VP1 protein in the CHO cell were detected by using immunohistochemistry and Western-blot,respectively. Enzyme digestion of PCR products and sequencing showed that the recombinant plasmid pEGFP-Cl-VPl was successfully constructed. RT-PCR result showed that the VP1 gene was tran-scripted in the CHO cell. Immunohistochemistry result showed that the expressed VP1 protein was mainly distributed in the membrane of CHO cells, and a little in cytoplasm. Western-blot showed that the expressed VP1 protein could bind to rabbit anti-EMCV antibody,indicating the immunoreactivity of recombinant VP1 protein.%为构建脑心肌炎病毒(EMCV) VP1基因的真核表达载体,并在CHO细胞中进行表达,通过RT-PCR扩增目的基因,酶切连接后将VP1基因克隆入真核表达载体pEGFP-C1中,通过脂质体法转染重组质粒pEGFP-C1-VP1,提取CHO细胞的总RNA,用RT-PCR检测目的基因的转录情况,并利用免疫组织化学和Western-blot分析检测目的基因表达情况、VP1蛋白在细胞中的定位及其抗原性.PCR、酶切验证及测序结果证实,重组质粒构建成功,绿色荧光蛋白正常表达.提取试验组细胞的总RNA,进行RT-PCR,在900 bp处扩增出了目的条带.免疫组织化学结果显示,VP1主要分布在CHO细胞的细胞膜中,少量存在于胞浆中.Western-blot分析结果证实,VP1能与兔抗EMCV抗体特异性结合.表明VP1蛋白在CHO细胞中表达良好,表达的蛋白具有反应原性.

  2. Expression of eukaryotic polypeptides in chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. BAGEL3: Automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides.

    Science.gov (United States)

    van Heel, Auke J; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P

    2013-07-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl.

  4. Comparison of eukaryotic phytobenthic community composition in a polluted river by partial 18S rRNA gene cloning and sequencing.

    Science.gov (United States)

    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.

  5. A New Method for Identification of Eukaryotic Gene Splice Sites%一种新的真核基因剪接位点识别方法

    Institute of Scientific and Technical Information of China (English)

    王科俊; 吕俊杰; 冯伟兴; 王鑫; 贺波

    2011-01-01

    剪接位点识别是基因组分析的关键步骤.为提高真核基因剪接位点识别的精度,提出一种融合多种信息的方法.在采用序列信息与剪接位点信号信息的基础上,增加剪接调控元件信息,并引入结构信息,针对供体位点与受体位点的不同特点,为其建立不同的识别模型.实验结果表明:该方法对剪接位点的识别具有较好的效果,其识别精度可达95%以上.%Splicing site recognition is the key step in the genome analysis. To improve the identification accuracy of eukary otic gene splicing sites, a variety of information fusion recognition method of splicing sites is proposed. Based on the using sequence information and splicing site signal information, we increased splicing regulatory element information, and proposed the structure information. By analyzing the different characteristics of donor sites and acceptor sites,donor sites identification signal model, acceptor sites identification signal model,donor sites identification sequence model, acceptor sites identification sequence model were built respectively. Our results show that the accuracy of splice site recognition is greater than 95 %, suggesting that the method has great potential to achieve a good performance for splice sites identification.

  6. Expression and construction of eukaryotic expression vector of porcine Mx1 gene%猪Mx1基因真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    毕峻龙; 沈学文; 李文贵; 舒相华; 杨贵树; 尹革芬

    2011-01-01

    The aim of this study was to constructe the eukaryotic expression vector of porcine Mx1 gene and express the protein effectively.The open reading frame(ORF) fragment of Mx1 gene was cloned according to the sequences in Genbank,then the pVa-Mx1-ORF expression vector was constructed by linking the ORF fragment and pVax1 vector.The Mx1 protein was expressed by transforming the pVa-Mx1-ORF vector into HEK-293 and BHK-21 cells and the expression products were identified using RT-PCR and indirect immunofluorescence assay.The results revealed that the Mx1 protein was expressed successfully in HEK-293 and BHK-21 cells.This study established the foundation for research on the mechanism of Mx1 protein anti-viral infection in vitro.%根据GenBank中猪Mx1基因序列设计并合成引物,对Mx1基因ORF区全长1 992个碱基进行扩增。将扩增片段与pVax1载体连接,构建pVa-Mx1-ORF真核表达载体。将构建的表达载体导入BHK-21和HEK-293细胞进行表达,用RT-PCR和间接免疫荧光对表达产物进行鉴定。结果表明,所构建的pVa-Mx1-ORF表达载体在BHK-21和HEK-293细胞中成功表达。

  7. Eukaryotic expression of bovine Neospora caninumMAG1 gene in Vero cells%牛源犬新孢子虫MAG1基因的克隆及在Vero细胞中表达

    Institute of Scientific and Technical Information of China (English)

    焦石; 贾立军; 刘明明; 黄国明; 张守发

    2011-01-01

    为了解牛源犬新孢子虫MAG1基因的生物学特性,本实验应用PCR技术扩增牛源犬新孢子虫MAG1基因,构建真核表达重组质粒pVAX-MAG1,将鉴定正确的pVAX-MAG1重组质粒转染Vero细胞,应用间接荧光检测方法(1FA)和western blot技术检测MAG1基因在Vero细胞中的表达.结果显示,扩增的牛源犬新孢子虫MAG1基因长度为1047bp,与GenBank中登录的MAG1 (EF580924.1)核苷酸序列同源性为99%,IFA检测MAG1基因在Vero细胞中获得瞬时表达,western blot分析表达蛋白的分子量为39 ku,具有较好的反应原性.本实验为新孢子虫病核酸疫苗与诊断试剂盒的研究奠定了基础.%To inestigate the biological characteristics of bovine Neospora caninum MAGI gene, the gene was amplified by PCR and inserted into eukaryotic expression vector. The resultant recombinant plasmid of pVAX-MAG 1 was transfected into Vero cells and the expression of MAGI was detected by indirect immunofluorescent assay, and western blot showed that the molecular weight of the protein was 39 ku and the protein had a positve reaction with anti MAGI serum. The results could be useful to DNA vaccine development.

  8. 湖羊MyoC基因CDS的克隆及其真核表达载体的构建%Cloning Based on Overlapping PCR and Construction of Eukaryotic Expression Vector of MyoG Gene of Hu Sheep

    Institute of Scientific and Technical Information of China (English)

    许峰; 秦玉蓉; 阴彦辉; 张振韬; 宋正海; 范广习; 高波; 李碧春

    2011-01-01

    Three exons of myogenin (MyoG)gene of Hu Sheep were amplified using three pairs of primers designed according to the MyoG gene sequence of Boer goat in GenBank, then linked by overlapping PCR, and inserted into the eukaryotic expression vector pcDNA3.0. NIH-3T3 cell was transfected with the constructed recombinant plasmid pcDNA3.0-MyoG, the expression of MyoG was detected by RT-PCR. The results showed that CDS of MyoG was successfully cloned in Hu sheep. Enzyme, PCR, RT-PCR analysis indicated that the recombinant plasmid pcDNA3. 0-MyoG was successfully constructed, which laid a foundation of MyoG in vivo and in vitro and its specific biological activity research.%参阅GenBank发表的波尔山羊肌细胞生成素(MyoC)基因序列,设计3对具有互补末端的特异性引物,分别扩增出湖羊MyoC基因的3个外显子,利用重叠PCR技术,将此3个外显子连接,并构建成重组质粒pcDNA3.0-MyoG,转染NIH-3T3细胞,RT-PCR鉴定.结果表明:成功克隆了湖羊MyoG基因的CDS,在离体细胞中MyoG基因发生了转录,这为进一步研究MyoG基因的体内外表达及生物学的活性奠定了基础.

  9. Eukaryotic expression of nucleocapsid protein gene from bovine parainfluenza virus type 3%牛副流感病毒3型核衣壳蛋白基因的真核表达

    Institute of Scientific and Technical Information of China (English)

    师新川; 温永俊; 王凤雪; 王炜; 王建科; 程世鹏; 武华

    2012-01-01

    The aim of this study is to construct an eukaryotic expression plasmid for nucleocapsid protein ( N) gene of bovine parainfluenza virus type 3 ( BPIV-3 ). According to the complete sequence of BPIV-3 NM09 strain ( GenBank accession: JQ063064 ) , a pair of primers was designed and synthesized. The N gene of BPIV3 was amplified by reverse transcription polymerase chain reaction (RT-PCR). The N gene was cloned into pcDNA3. 1 (+) vector using Nhel and Notl, and thus the expression plasmid pcDNA3. 1-N was successfully constructed. Then pcDNA3. 1-N was transfected into BSR cell using lipoplast mediate method and positive results were demonstrated by indirect im-munofluorescence assay (IFA) and RT-PCR. The results showed N gene of BPIV-3 was successfully expressed in BSR cell. The study provided fundamental information for the further study on the prevention of BPIV3 infection and new vaccine development%根据牛副流感病毒3型(BPIV-3)内蒙09株(NM09)全基因组序列(GenBank登录号:JQ063064)设计特异性引物,利用RT-PCR方法扩增出牛副流感病毒3型分离株的核衣壳蛋白(N)基因,通过NheI和NotI限制性内切酶位点亚克隆至真核表达载体pcDNA3.1/Zeo(+),获得真核重组质粒pcDNA3.1-N.采用Superfect转染试剂将重组质粒转染至BSR细胞中,转染后的BSR细胞经间接免疫荧光试验和RT-PCR方法检测,重组质粒pcDNA3.1-N在BSR细胞中能正确表达N蛋白.本研究结果为BPIV-3新型疫苗的研制奠定基础.

  10. Eukaryotic vs. prokaryotic chemosensory systems.

    Science.gov (United States)

    Sbarbati, Andrea; Merigo, Flavia; Osculati, Francesco

    2010-04-01

    In the last decades, microbiologists demonstrated that microorganisms possess chemosensory capabilities and communicate with each other via chemical signals. In parallel, it was demonstrated that solitary eukaryotic chemosensory cells are diffusely located on the mucosae of digestive and respiratory apparatuses. It is now evident that on the mucosal surfaces of vertebrates, two chemoreceptorial systems (i.e. eukaryotic and prokaryotic) coexist in a common microenvironment. To date, it is not known if the two chemosensory systems reciprocally interact and compete for detection of chemical cues. This appears to be a fruitful field of study and future researches must consider that the mucosal epithelia possess more chemosensory capabilities than previously supposed.

  11. Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing

    Directory of Open Access Journals (Sweden)

    Miri eMichaeli

    2012-12-01

    Full Text Available High throughput sequencing (HTS yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig genes, which are variable and often highly mutated. This paper describes Ig-HTS-Cleaner (Ig High Throughput Sequencing Cleaner, a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig-Indel-Identifier (Ig Insertion – Deletion Identifier, a program for identifying legitimate and artifact insertions and/or deletions (indels. Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.

  12. Mutation Profile of B-Raf Gene Analyzed by fully Automated System and Clinical Features in Japanese Melanoma Patients.

    Science.gov (United States)

    Ide, Masaru; Koba, Shinichi; Sueoka-Aragane, Naoko; Sato, Akemi; Nagano, Yuri; Inoue, Takuya; Misago, Noriyuki; Narisawa, Yutaka; Kimura, Shinya; Sueoka, Eisaburo

    2017-01-01

    BRAF gene mutations have been observed in 30-50 % of malignant melanoma patients. Recent development of therapeutic intervention using BRAF inhibitors requires an accurate and rapid detection system for BRAF mutations. In addition, the clinical characteristics of the melanoma associated with BRAF mutations in Japanese patients have not been investigated on a large scale evaluation. We recently established quenching probe system (QP) for detection of an activating BRAF mutation, V600E and evaluated 113 melanoma samples diagnosed in Saga University Hospital from 1982 to 2011. The QP system includes fully automated genotyping, based on analysis of the probe DNA melting curve, which binds the target mutated site using a fluorescent guanine quenched probe. BRAF mutations were detected in 54 of 115 (47 %) including 51 of V600E and 3 of V600 K in Japanese melanoma cases. Among clinical subtypes of melanoma, nodular melanoma showed high frequency (12 of 15; 80 %) of mutation followed by superficial spreading melanoma (13 of 26; 50 %). The QP system is a simple and sensitive method to determine BRAF V600E mutation, and will be useful tool for patient-oriented therapy with BRAF inhibitors.

  13. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  14. Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth

    Science.gov (United States)

    Milias-Argeitis, Andreas; Rullan, Marc; Aoki, Stephanie K.; Buchmann, Peter; Khammash, Mustafa

    2016-01-01

    Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology. PMID:27562138

  15. Clasificación automática de los estados de desarrollo del arroz a partir de imágenes de RADARSAT-2

    OpenAIRE

    Quiñones Serrano, Jorge Arturo

    2011-01-01

    Las imágenes de radar se utilizan tanto para la identificación, como para el seguimiento del crecimiento y la medición de las superficies destinadas a los diferentes cultivos. En este trabajo, utilizamos una imagen de radar capturada por el satélite RADARSAT-2 en el mes de febrero de 2009, para implementar un sistema de clasificación automática de los estados de desarrollo del cultivo de arroz. Debido a que las imágenes presentan un ruido de moteado característico, en la primera parte del pro...

  16. Phylogenomic analysis of the cystatin superfamily in eukaryotes and prokaryotes

    Directory of Open Access Journals (Sweden)

    Turk Vito

    2009-11-01

    Full Text Available Abstract Background The cystatin superfamily comprises cysteine protease inhibitors that play key regulatory roles in protein degradation processes. Although they have been the subject of many studies, little is known about their genesis, evolution and functional diversification. Our aim has been to obtain a comprehensive insight into their origin, distribution, diversity, evolution and classification in Eukaryota, Bacteria and Archaea. Results We have identified in silico the full complement of the cystatin superfamily in more than 2100 prokaryotic and eukaryotic genomes. The analysis of numerous eukaryotic genomes has provided strong evidence for the emergence of this superfamily in the ancestor of eukaryotes. The progenitor of this superfamily was most probably intracellular and lacked a signal peptide and disulfide bridges, much like the extant Giardia cystatin. A primordial gene duplication produced two ancestral eukaryotic lineages, cystatins and stefins. While stefins remain encoded by a single or a small number of genes throughout the eukaryotes, the cystatins have undergone a more complex and dynamic evolution through numerous gene and domain duplications. In the cystatin superfamily we discovered twenty vertebrate-specific and three angiosperm-specific orthologous families, indicating that functional diversification has occurred only in multicellular eukaryotes. In vertebrate orthologous families, the prevailing trends were loss of the ancestral inhibitory activity and acquisition of novel functions in innate immunity. Bacterial cystatins and stefins may be emergency inhibitors that enable survival of bacteria in the host, defending them from the host's proteolytic activity. Conclusion This study challenges the current view on the classification, origin and evolution of the cystatin superfamily and provides valuable insights into their functional diversification. The findings of this comprehensive study provide guides for future

  17. Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite.

    Science.gov (United States)

    Nakagawa, Takahito; Maruyama, Kohei; Takeyama, Haruko; Matsunaga, Tadashi

    2007-04-15

    The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.

  18. Warehouse automation

    OpenAIRE

    Pogačnik, Jure

    2017-01-01

    An automated high bay warehouse is commonly used for storing large number of material with a high throughput. In an automated warehouse pallet movements are mainly performed by a number of automated devices like conveyors systems, trolleys, and stacker cranes. From the introduction of the material to the automated warehouse system to its dispatch the system requires no operator input or intervention since all material movements are done automatically. This allows the automated warehouse to op...

  19. Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers.

    Science.gov (United States)

    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.

  20. What Entamoeba histolytica and Giardia lamblia tell us about the evolution of eukaryotic diversity

    Indian Academy of Sciences (India)

    J Samuelson

    2002-11-01

    Entamoeba histolytica and Giardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions, early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.

  1. RNA Export through the NPC in Eukaryotes.

    Science.gov (United States)

    Okamura, Masumi; Inose, Haruko; Masuda, Seiji

    2015-03-20

    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.

  2. Censusing marine eukaryotic diversity in the twenty-first century.

    Science.gov (United States)

    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'.

  3. Censusing marine eukaryotic diversity in the twenty-first century

    Science.gov (United States)

    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

  4. Construction and Function Identification of Eukaryotic Plasmid Carrying MouseFoxc2 Gene%小鼠Foxc2质粒载体的构建及功能鉴定

    Institute of Scientific and Technical Information of China (English)

    赵霞; 景旭斌; 刘静; 赵涛涛; 杨海丽; 孙超

    2012-01-01

    In order to construct eukaryotic plasmid carrying mouse suppressors of Foxc2 gene, 3T3-L1 preadipocytes were transferred by Lipofection 2000 to preliminary study the lipid metabolism of Foxc2 .Total RNA was extracted from mouse subcutaneous fat, plasmids of pMD18-T- Foxc2 and pcDNA3. 1- Foxc2 was successfully constructed after PCR and TA cloning. Plasmid pcDNA 3.1-Foxc2 was confirmed by restriction digestion and Foxc2 gene sequencing which was consistent with that of NCBI gene bank. The recombinant plasmid pcDNA3. 1-Foxc2 was transfected into 3T3-L1 cell line. The non-transfected 3T3-L1 cells and 3T3-L1 cells transfected with empty vector were served as controls. The mRNA and protein of Foxc2 gene were identified by real-time PCR and western blot analysis. Meanwhile, the expressions of FAS, ATGL, HSL and PPARγ were detected. The result showed that the mRN A and protein of Foxc2 expression in recombinant pcDNA3. 1-Foxc2 group were significantly higher than in un-transfected group and empty vector group. In 3T3-L1 preadipocytes, overexpression Foxc2 significantly reduced the expression of FAS and significantly increased the expressions of HSL and PPARγ , but had little effect on ATGL, which indicated that Foxc2 functioned to inhibit fat deposition. These results provide the basis for further study of Foxc2 functioned in signaling pathway of fat metabolism in mice 3T3-L1 adipocytes.%构建pcDNA3.1-Foxc2真核表达载体,并以脂质体转染3T3-L1前体脂肪细胞,探究Foxc2对脂代谢的影响.取孕后15 d小鼠腹部脂肪组织提取总RNA,用PCR扩增Foxc2全长序列并连接至pMD18-T载体,测序正确后克隆至pcDNA3.1(+)真核表达载体,成功构建pcDNA3.1-Foxc2重组质粒载体.重组质粒转染3T3-L1前体脂肪细胞系,以未转染和空载体转染作为对照,通过RT-PCR和Western blotting法检测Foxc2核酸及蛋白表达,同时检测3T3-L1前体脂肪细胞中脂代谢相关基因FAS、ATGL、HSL及PPARγ的表达.结果显示,重

  5. Construction of Eukaryotic Expression Vector with Rana Antimicrobial Peptides Gene Temporin-lCEa%中国林蛙抗菌肽Temporin-1CEa基因的真核表达载体构建。

    Institute of Scientific and Technical Information of China (English)

    张志崇; 王春生; 张秋婷; 朴善花; 苗向阳; 安铁洙

    2012-01-01

    In order to establish a method to get a large number of antimicrobial peptides from Rana chensinensis,a series of experiments were conducted as follows.According to Chinese frog skin antimicrobial peptides Temporin-1CEa gene mRNA sequence(EU624139) in GenBank,a pair of specific primers were designed and cDNA was obtained from Chinese forest frog skin RNA by reverse transcription.Temporin-1CEa gene coding sequence was amplified using the cDNA,and linked with pEASY-T3 cloning vector.The GFP gene was inserted into the recombinant plasmid Tem-T3 by molecular methods.The Tem-GFP fragment was linked with eukaryotic expression vector pcDNA3.1,and Tem-GFP-pcDNA3.1 recombinant plasmid was achieved finally.Using of lipid infection method,the plasmids were transfected into sheep fibroblast cells,the green fluorescence was observed under a fluorescence microscope after 48 h.qPCR data showed that Tem-GFP fusion protein expression level of transfected Tem-GFP-pcDNA3.1 sheep fibroblasts increased about 300 folds than that of the control group.This study supplied the technical basis for developing mammary gland bioreactor of expressing Temporin-1CEa gene.%为了建立大量获取中国林蛙抗菌肽的方法,根据GenBank中的中国林蛙皮肤抗菌肽Temporin-1CEa基因的mRNA序列(EU624139)设计一对特异性引物,以提取的中国林蛙皮肤总RNA反转录出的cDNA为模板,将扩增的编码序列与pEASY-T3克隆载体连接获得Tem-T3;利用酶切、连接等分子生物学手段,将GFP基因连入Tem-T3克隆载体,再经酶切获得Tem-GFP片段,并插入真核表达载体pcDNA3.1,最终得到Tem-GFP-pcD-NA3.1重组质粒;利用脂质体转染法将该质粒转入绵羊成纤维细胞,48 h后可在荧光倒置显微镜下观察到GFP的绿色荧光表达;qPCR数据分析显示,与对照组相比,转染Tem-GFP-pcDNA3.1的绵羊成纤维细胞中融合蛋白Tem-GFP的表达量可提高约300倍。本研究为构建Temporin-1CEa基因山羊乳腺特异表达载体提供依据。

  6. Defensins: antifungal lessons from eukaryotes

    Directory of Open Access Journals (Sweden)

    Patrícia M. Silva

    2014-03-01

    Full Text Available Over the last years, antimicrobial peptides (AMPs have been the focus of intense research towards 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, Plantæ 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 citotoxicity are also reviewed.

  7. Generation and Expression of Recombinant Eukaryotic Expression Plasmids of PAX3 Gene and Its Significance%PAX3基因重组真核细胞表达质粒的构建、表达及意义

    Institute of Scientific and Technical Information of China (English)

    张华; 李家大; 罗浑金; 陈红胜; 梅凌云; 贺楚峰; 冯永

    2014-01-01

    目的:通过构建PAX3基因及其突变体表达质粒初步研究其外源性表达和定位表达,为研究Waar-denburg综合征(Waardenburg syndrome ,WS)发病机制提供实验基础。方法通过分子克隆技术双酶切pECE -PAX3和pcDNA3.0-HA后连接构建PAX3基因重组真核细胞表达质粒pcDNA3.0-PAX3-HA ,以其为模板分别构建PAX3基因新发突变H80D和H186fs表达质粒pcDNA3.0-H80D-HA和pcDNA3.0-H186fs-HA , DNA测序鉴定。将PAX3、H80D和H186fs表达质粒分别瞬时转染小鼠胚胎成纤维细胞(NIH3T3细胞),采用Western blot和细胞免疫荧光法分别检测和观察野生PAX3蛋白和突变 H80D、H186fs蛋白在NIH3T3细胞中的表达和分布。结果 PAX3及其突变体 H80D和H186fs表达质粒经DNA测序鉴定序列正确,三者在NIH3T3细胞中正确表达,H80D与PAX3仅在细胞核中分布,H186fs在细胞质与细胞核中均有分布。结论本研究成功构建了PAX3基因及其突变体真核细胞表达质粒,突变对PAX3蛋白的亚细胞定位产生影响,为在体外实验进一步研究国人PAX3基因突变致WS发病的分子机制奠定了实验基础。%Objective To study exogenous expression and subcellular localization of wild type (WT ) and mu-tant PAX3 proteins in vitro by generating their expression plasmids for further study of pathogenesis of Waarden-burg syndrome (WS) .Methods The plasmids pECE-PAX3 and pcDNA3 .0-HA were ligased after they were cut by double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pcDNA3 .0-PAX3-HA ,which was as a template to generate expression plasmids pcDNA 3 .0 -H80D -HA and pcDNA3 .0-H186fs-HA of novel mutations H80D and H186fs of PAX3 gene .All constructs were verified by di-rect nucleotide sequencing .NIH3T3 cells were transfected transiently with the expression plasmids of PAX3 ,H80D and H186fs respectively .The exogenous expression of WT PAX3 protein and mutant

  8. Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L+ T Cells for Manufacturing of Gene Therapy Medicinal Products

    Science.gov (United States)

    Priesner, Christoph; Aleksandrova, Krasimira; Esser, Ruth; Mockel-Tenbrinck, Nadine; Leise, Jana; Drechsel, Katharina; Marburger, Michael; Quaiser, Andrea; Goudeva, Lilia; Arseniev, Lubomir; Kaiser, Andrew D.; Glienke, Wolfgang; Koehl, Ulrike

    2016-01-01

    Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies. PMID:27562135

  9. CGMIM: Automated text-mining of Online Mendelian Inheritance in Man (OMIM to identify genetically-associated cancers and candidate genes

    Directory of Open Access Journals (Sweden)

    Jones Steven

    2005-03-01

    Full Text Available Abstract Background Online Mendelian Inheritance in Man (OMIM is a computerized database of information about genes and heritable traits in human populations, based on information reported in the scientific literature. Our objective was to establish an automated text-mining system for OMIM that will identify genetically-related cancers and cancer-related genes. We developed the computer program CGMIM to search for entries in OMIM that are related to one or more cancer types. We performed manual searches of OMIM to verify the program results. Results In the OMIM database on September 30, 2004, CGMIM identified 1943 genes related to cancer. BRCA2 (OMIM *164757, BRAF (OMIM *164757 and CDKN2A (OMIM *600160 were each related to 14 types of cancer. There were 45 genes related to cancer of the esophagus, 121 genes related to cancer of the stomach, and 21 genes related to both. Analysis of CGMIM results indicate that fewer than three gene entries in OMIM should mention both, and the more than seven-fold discrepancy suggests cancers of the esophagus and stomach are more genetically related than current literature suggests. Conclusion CGMIM identifies genetically-related cancers and cancer-related genes. In several ways, cancers with shared genetic etiology are anticipated to lead to further etiologic hypotheses and advances regarding environmental agents. CGMIM results are posted monthly and the source code can be obtained free of charge from the BC Cancer Research Centre website http://www.bccrc.ca/ccr/CGMIM.

  10. EUKARYOTIC EXPRESSION AND ANTIVIRAL ACTIVITY OF PORCINE MYXOVIRUS RESISTANCE GENE (Mx1)%猪Mx1基因的克隆表达及抗病毒活性研究

    Institute of Scientific and Technical Information of China (English)

    刘玉洁; 赵协; 张廷虹; 曹广明; 扬志昆

    2014-01-01

    本文应用(RT-PCR)技术,从猪瘟弱毒疫苗和Poly:IC共刺激7 h的PK15细胞的cDNA中扩增出编码Mx1蛋白的全长基因,并通过引物设计填补了PK(15)细胞系Mx1 cDNA序列中3'端11bp的缺失,成功获得Mx1完整基因的克隆。构建了重组表达质粒pRetroQ-sMx1,转染HEK293T细胞并表达Mx1蛋白,利用微量细胞病变抑制法测定其抗病毒活性。双酶切鉴定和核酸序列测定证实pRetroQ-sMx1真核表达质粒构建成功,转染HEK 293T细胞后,能够检测到绿色荧光,Western blot证实为目的蛋白。微量细胞病变抑制法测定重组蛋白具有一定的抗水疱性口炎病毒及猪繁殖与呼吸综合征病毒的活性。重组Mx1具有一定抗病毒生物功能,为进一步研究重组Mx1蛋白的活性以及Mx1抗病毒药物奠定了基础。%The present study was to clone Myxovirus resistance protein Mx1 for eukaryotic expression and detect its antiviral activity. The RNA coding for Mx1 was extracted from PK15 cells that were stimulated for 7 hours with swine fever vaccine and Poly real:IC. Subsequently, full-length Mx1 gene was amplified in RT-PCR using primers designed to rescue 11 bp missing in PK 15 cell line. The recombinant expression plasmid pRetroQ-sMx1 was constructed and used to transfect HEK293T cells for expression. Success of Mx1 expression in HEK293T cells was confirmed through double enzyme identification, determination of nucleic acid sequence, detection of green fluorescence and Western blot. Cytopathic effect inhibition assay was used to measure antiviral activity of expressed Mx1.

  11. Construction of Eukaryotic Expression Vector and Sequence Analysis of Antimicrobial Peptide Gene Shiva 1a%抗菌肽Shiva1a基因真核表达载体的构建及序列分析

    Institute of Scientific and Technical Information of China (English)

    宫晓炜; 郑福英; 蔺国珍; 曹小安; 王光华; 周继章; 才学鹏

    2011-01-01

    为了探讨天蚕类抗菌肽在动物早期抗感染过程中的作用机理,本研究以Shiva 1a基因的成熟肽为模板设计4条引物,利用重叠延伸PCR技术获得目的基因,并在C端添加6×His的标签.将此序列与真核表达载体pIRES2 -EGFP进行重组,构建pIRES2-EGFP-Shiva 1a重组表达质粒,对重组质粒进行酶切和测序鉴定后,采用阳离子脂质体转染将重组质粒转染到CHO-K1细胞,荧光显微镜观察其表达情况.通过生物信息学软件对抗菌肽Shiva 1a的二级结构和三级结构进行预测分析.其结果为进一步研究Shiva 1a的抗菌活性和在动物抗病育种方面的应用奠定了基础.%To explore the effect mechanism of cecropin-class lytic peptide at early stage of infection. The mat peptide of Shiva la was amplified by overlap extension PCR and the C-terminus contained 6×His-marker. The gene sequence were recombi-nant with eukaryotic expression vector pIRES2-EGFP. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmid pIRES2-EGFP-Shiva la was transfected into CHO-K1 cells by liposomes. The expression of the the recombinant plasmid pIRES2-EGFP-Shiva 1a was observed by fluorescence microscope. At the same time, the secondary structure and 3D structure were predicted by bioinformatics tools. The results lay the foundation in research of antimicrobial activities and applications of the antimicrobial peptide Shiva la in breeding for disease resistance of animals.

  12. EuCAP, a Eukaryotic Community Annotation Package, and its application to the rice genome

    Directory of Open Access Journals (Sweden)

    Hamilton John P

    2007-10-01

    Full Text Available Abstract Background Despite the improvements of tools for automated annotation of genome sequences, manual curation at the structural and functional level can provide an increased level of refinement to genome annotation. The Institute for Genomic Research Rice Genome Annotation (hereafter named the Osa1 Genome Annotation is the product of an automated pipeline and, for this reason, will benefit from the input of biologists with expertise in rice and/or particular gene families. Leveraging knowledge from a dispersed community of scientists is a demonstrated way of improving a genome annotation. This requires tools that facilitate 1 the submission of gene annotation to an annotation project, 2 the review of the submitted models by project annotators, and 3 the incorporation of the submitted models in the ongoing annotation effort. Results We have developed the Eukaryotic Community Annotation Package (EuCAP, an annotation tool, and have applied it to the rice genome. The primary level of curation by community annotators (CA has been the annotation of gene families. Annotation can be submitted by email or through the EuCAP Web Tool. The CA models are aligned to the rice pseudomolecules and the coordinates of these alignments, along with functional annotation, are stored in the MySQL EuCAP Gene Model database. Web pages displaying the alignments of the CA models to the Osa1 Genome models are automatically generated from the EuCAP Gene Model database. The alignments are reviewed by the project annotators (PAs in the context of experimental evidence. Upon approval by the PAs, the CA models, along with the corresponding functional annotations, are integrated into the Osa1 Genome Annotation. The CA annotations, grouped by family, are displayed on the Community Annotation pages of the project website http://rice.tigr.org, as well as in the Community Annotation track of the Genome Browser. Conclusion We have applied EuCAP to rice. As of July 2007, the

  13. Evolutionary position of breviate amoebae and the primary eukaryote divergence.

    Science.gov (United States)

    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.

  14. Accounting Automation

    OpenAIRE

    Laynebaril1

    2017-01-01

    Accounting Automation   Click Link Below To Buy:   http://hwcampus.com/shop/accounting-automation/  Or Visit www.hwcampus.com Accounting Automation” Please respond to the following: Imagine you are a consultant hired to convert a manual accounting system to an automated system. Suggest the key advantages and disadvantages of automating a manual accounting system. Identify the most important step in the conversion process. Provide a rationale for your response. ...

  15. Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

    DEFF Research Database (Denmark)

    Møller, Annette; Asp, Torben; Holm, Preben Bach

    2008-01-01

    Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps...... exclusive for the secretory pathway of eukaryotes by combining the identification of lineage-specific genes with phylogenetic evolution of common genes. Sequences of P5 ATPases, which are regarded to be cation pumps in the endoplasmic reticulum (ER), were identified in all eukaryotic lineages but not in any...

  16. Construction of Eukaryotic Coexpression Vector Containing Mycobacterium Tuberculosis Heat Shock Protein 70 and Herpes Simplex Virus Thymidine Kinase Genes%mtHSP70/HSV-TK双基因真核共表达质粒的构建

    Institute of Scientific and Technical Information of China (English)

    曾曙光; 刘启才; 王素文; 徐平平; 章锦才; 张积仁

    2011-01-01

    目的 构建结核杆菌热休克蛋白70(mycobacterium tuberculosis heat-shock proteins 70,mtHSP70)和自杀基因单纯疱疹病毒-胸苷激酶(herpes simplex virus-thymidinekinase,HSV-TK)双基因真核共表达质粒pCMV-mtHSP70-IRES-TK.方法 登录Genbank查询HSV-TK和mtHSP70基因mRNA序列,应用引物设计软件DNA club分别设计扩增HSV-TK和mtHSP70基因全长cDNA的特异引物.以pcDNA3-TK质粒或mtHSP70质粒DNA为模板,分别采用各自的引物,用prime STAR HS DNA Polymerase进行聚合酶链反应(polymerase chain reaction,PCR),获得HSV-TK和mtHSP70基因cDNA片断,连接到T载体pMD18-T上,转化大肠杆菌TG1后用HSV-TK和mtHSP70基因的特异引物进行菌落PCR,确定含有阳性mtHSP70或HSV-TK重组质粒并将阳性pMD18T-TK重组质粒和pMD18T-mtHSP70重组质粒进行上、下游测序,测序结果与基因序列进行同源比较.选定正确的质粒用于HSV-TK和mtHSP70基因的亚克隆,构建质粒PCMV-mtHSP70-IRES-TK,所得阳性克隆摇菌后少量提取质粒,分别用NotⅠ单酶切及EcoRⅠ和XhoⅠ双酶切进行酶切鉴定.结果 构建的pCMV-mtHSP70-IRES-TK质粒分别用NotⅠ单酶切及EcoRⅠ/XhoⅠ双酶切进行酶切鉴定,酶切产物电泳见特异酶切图谱,确定为重组质粒pCMV-mtHSP70-IRES-TK.结论 本实验为后续研究mtHSP70/HSV-TK双基因的协同抗肿瘤作用奠定了实验基础.%Objective To construct an eukaryotic plasmid PCMV-mtHSP70-IRES-TK that contains and expresses both mycobacterium tuberculosis heat shock protein 70 (mtHSP70) gene and suicide gene HSV-TK for the experiments. Methods Login on genbank and search for mRNA sequences of HSV-TK and mtHSP70 genes. Specific primers for the amplification of full length cDNA of HSV-TK and mtHSP70 were designed by primer designing software DNA club. pcDNA3-TK plasmid and mtHSP70 plasmid DNA were used as template. PCR was performed with respective primers and prime STAR HS DNA polymerase, cDNA segments of HSV-TK and mtHSP70

  17. Home Automation

    OpenAIRE

    Ahmed, Zeeshan

    2010-01-01

    In this paper I briefly discuss the importance of home automation system. Going in to the details I briefly present a real time designed and implemented software and hardware oriented house automation research project, capable of automating house's electricity and providing a security system to detect the presence of unexpected behavior.

  18. Non-coding RNAs: the architects of eukaryotic complexity.

    Science.gov (United States)

    Mattick, J S

    2001-11-01

    Around 98% of all transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.

  19. Open Questions on the Origin of Eukaryotes.

    Science.gov (United States)

    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.

  20. BEACON: automated tool for Bacterial GEnome Annotation ComparisON

    KAUST Repository

    Kalkatawi, Manal Matoq Saeed

    2015-08-18

    Background Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs). Results The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced. Conclusions We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/

  1. DNA polymerase zeta (polζ) in higher eukaryotes

    Institute of Scientific and Technical Information of China (English)

    Gregory N Gan; John P Wittschieben; Birgitte φ Wittschieben; Richard D Wood

    2008-01-01

    Most current knowledge about DNA polymerase zeta (pol ζ) comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where polζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Rev1. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already appar-ent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genuine instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7(MAD2B) in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair ofinterstrand DNA erosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent devel-opments in these areas.

  2. A high-affinity molybdate transporter in eukaryotes.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  3. Detección automática de NEOs en imágenes CCD utilizando la transformada de Hough

    Science.gov (United States)

    Ruétalo, M.; Tancredi, G.

    El interés y la dedicación por los objetos que se acercan a la órbita de la Tierra (NEOs) ha aumentado considerablemente en los últimos años, tanto que se han iniciado varias campañas de búsqueda sistemática para aumentar la población identificada de éstos. El uso de placas fotográficas e identificación visual está siendo sustituído, progresivamente, por el uso de cámaras CCD y paquetes de detección automática de los objetos en las imágenes digitales. Una parte muy importante para la implementación exitosa de un programa automatizado de detección de este tipo es el desarrollo de algoritmos capaces de identificar objetos de baja relación señal-ruido y con requerimientos computacionales no elevados. En el presente trabajo proponemos la utilización de la transformada de Hough (utilizada en algunas áreas de visión artificial) para detectar automáticamente trazas, aproximadamente rectilíneas y de baja relación señal-ruido, en imágenes CCD. Desarrollamos una primera implementación de un algoritmo basado en ésta y lo probamos con una serie de imágenes reales conteniendo trazas con picos de señales de entre ~1 σ y ~3 σ por encima del nivel del ruido de fondo. El algoritmo detecta, sin inconvenientes, la mayoría de los casos y en tiempos razonablemente adecuados.

  4. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V.; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  5. Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

    Science.gov (United States)

    Huberman, Eliezer; Baccam, Mekhine J.

    2007-02-27

    The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

  6. Characterization of prokaryotic and eukaryotic promoters usinghidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Brunak, Søren

    1996-01-01

    that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma-70 and sigma-54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely......In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... have at the same time the ability to find clusters and the ability to model the sequential structure in the input data. This is highly relevant in situations where the variance in the data is high, as is the case for the subclass structure in for example promoter sequences....

  7. Characterization of prokaryotic and eukaryotic promoters using hidden Markov models

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, P.; Chauvin, Y.

    1996-01-01

    that bind to them. We find that HMMs trained on such subclasses of Escherichia coli promoters (specifically, the so-called sigma 70 and sigma 54 classes) give an excellent classification of unknown promoters with respect to sigma-class. HMMs trained on eukaryotic sequences from human genes also model nicely......In this paper we utilize hidden Markov models (HMMs) and information theory to analyze prokaryotic and eukaryotic promoters. We perform this analysis with special emphasis on the fact that promoters are divided into a number of different classes, depending on which polymerase-associated factors...... have at the same time the ability to find clusters and the ability to model the sequential structure in the input data. This is highly relevant in situations where the variance in the data is high, as is the case for the subclass structure in for example promoter sequences....

  8. Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton.

    Science.gov (United States)

    Cuvelier, Marie L; Allen, Andrew E; Monier, Adam; McCrow, John P; Messié, Monique; Tringe, Susannah G; Woyke, Tanja; Welsh, Rory M; Ishoey, Thomas; Lee, Jae-Hyeok; Binder, Brian J; DuPont, Chris L; Latasa, Mikel; Guigand, Cédric; Buck, Kurt R; Hilton, Jason; Thiagarajan, Mathangi; Caler, Elisabet; Read, Betsy; Lasken, Roger S; Chavez, Francisco P; Worden, Alexandra Z

    2010-08-17

    Among eukaryotes, four major phytoplankton lineages are responsible for marine photosynthesis; prymnesiophytes, alveolates, stramenopiles, and prasinophytes. Contributions by individual taxa, however, are not well known, and genomes have been analyzed from only the latter two lineages. Tiny "picoplanktonic" members of the prymnesiophyte lineage have long been inferred to be ecologically important but remain poorly characterized. Here, we examine pico-prymnesiophyte evolutionary history and ecology using cultivation-independent methods. 18S rRNA gene analysis showed pico-prymnesiophytes belonged to broadly distributed uncultivated taxa. Therefore, we used targeted metagenomics to analyze uncultured pico-prymnesiophytes sorted by flow cytometry from subtropical North Atlantic waters. The data reveal a composite nuclear-encoded gene repertoire with strong green-lineage affiliations, which contrasts with the evolutionary history indicated by the plastid genome. Measured pico-prymnesiophyte growth rates were rapid in this region, resulting in primary production contributions similar to the cyanobacterium Prochlorococcus. On average, pico-prymnesiophytes formed 25% of global picophytoplankton biomass, with differing contributions in five biogeographical provinces spanning tropical to subpolar systems. Elements likely contributing to success include high gene density and genes potentially involved in defense and nutrient uptake. Our findings have implications reaching beyond pico-prymnesiophytes, to the prasinophytes and stramenopiles. For example, prevalence of putative Ni-containing superoxide dismutases (SODs), instead of Fe-containing SODs, seems to be a common adaptation among eukaryotic phytoplankton for reducing Fe quotas in low-Fe modern oceans. Moreover, highly mosaic gene repertoires, although compositionally distinct for each major eukaryotic lineage, now seem to be an underlying facet of successful marine phytoplankton.

  9. Construction of eukaryotic vector of monkey B virus glycoprotein D gene and the gD gene expression%猕猴B病毒囊膜蛋白gD基因真核细胞表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    王新; 易思萌; 刘慧芳; 马凯; 范君文; 马雨楠; 游颖; 孙兆增

    2015-01-01

    Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.%目的:构建猕猴B病毒囊膜蛋白gD的真核表达载体,并且检测其在293T细胞内的表达情况。方法首先通过基因合成手段获得含B病毒gD蛋白的基因片段,在经由PstⅠ和NotⅠ双酶切后连接到pEGFP-N3载体,随后将构建的pEGFP-N3-GD重组质粒转染到人胚胎肾上皮细胞系293T细胞。再用Western blot检测所提蛋白其在细胞内的表达情况,并用激光共聚焦分析其在细胞内的表达定位情况。结果成功获得携带gD基因的阳性重组质粒pEGFP-N3-GD,且pEGFP-N3-GD重组质粒能在293T细胞的表面正常表达。结论利用真核表达系统,既能够在细胞表面产生B病毒gD蛋白的特异性重组抗原,而且可用于B检测抗原的制备。

  10. Pathogenic eukaryotes in gut microbiota of western lowland gorillas as revealed by molecular survey.

    Science.gov (United States)

    Hamad, Ibrahim; Keita, Mamadou B; Peeters, Martine; Delaporte, Eric; Raoult, Didier; Bittar, Fadi

    2014-09-18

    Although gorillas regarded as the largest extant species of primates and have a close phylogenetic relationship with humans, eukaryotic communities have not been previously studied in these populations. Herein, 35 eukaryotic primer sets targeting the 18S rRNA gene, internal transcribed spacer gene and other specific genes were used firstly to explore the eukaryotes in a fecal sample from a wild western lowland gorilla (Gorilla gorilla gorilla). Then specific real-time PCRs were achieved in additional 48 fecal samples from 21 individual gorillas to investigate the presence of human eukaryotic pathogens. In total, 1,572 clones were obtained and sequenced from the 15 cloning libraries, resulting in the retrieval of 87 eukaryotic species, including 52 fungi, 10 protozoa, 4 nematodes and 21 plant species, of which 52, 5, 2 and 21 species, respectively, have never before been described in gorillas. We also reported the occurrence of pathogenic fungi and parasites (i.e. Oesophagostomum bifurcum (86%), Necator americanus (43%), Candida tropicalis (81%) and other pathogenic fungi were identified). In conclusion, molecular techniques using multiple primer sets may offer an effective tool to study complex eukaryotic communities and to identify potential pathogens in the gastrointestinal tracts of primates.

  11. Automation or De-automation

    Science.gov (United States)

    Gorlach, Igor; Wessel, Oliver

    2008-09-01

    In the global automotive industry, for decades, vehicle manufacturers have continually increased the level of automation of production systems in order to be competitive. However, there is a new trend to decrease the level of automation, especially in final car assembly, for reasons of economy and flexibility. In this research, the final car assembly lines at three production sites of Volkswagen are analysed in order to determine the best level of automation for each, in terms of manufacturing costs, productivity, quality and flexibility. The case study is based on the methodology proposed by the Fraunhofer Institute. The results of the analysis indicate that fully automated assembly systems are not necessarily the best option in terms of cost, productivity and quality combined, which is attributed to high complexity of final car assembly systems; some de-automation is therefore recommended. On the other hand, the analysis shows that low automation can result in poor product quality due to reasons related to plant location, such as inadequate workers' skills, motivation, etc. Hence, the automation strategy should be formulated on the basis of analysis of all relevant aspects of the manufacturing process, such as costs, quality, productivity and flexibility in relation to the local context. A more balanced combination of automated and manual assembly operations provides better utilisation of equipment, reduces production costs and improves throughput.

  12. GEGEINTOOL: A Computer-Based Tool for Automated Analysis of Gene-Gene Interactions in Large Epidemiological Studies in Cardiovascular Genomics

    Directory of Open Access Journals (Sweden)

    Oscar Coltell

    2013-06-01

    Full Text Available Current methods of data analysis of gene-gene interactions in complex diseases, after taking into account environmental factors using traditional approaches, are inefficient. High-throughput methods of analysis in large scale studies including thousands of subjects and hundreds of SNPs should be implemented. We developed an integrative computer tool, GEGEINTOOL (GEne- GEne INTeraction tOOL, for large-scale analysis of gene-gene interactions, in human studies of complex diseases including a large number of subjects, SNPs, as well as environmental factors. That resource uses standard statistical packages (SPSS, etc. to build and fit the gene-gene interaction models by means of syntax scripts in predicting one or more continuous or dichotomic phenotypes. Codominant, dominant and recessive genetic interaction models including control for covariates are automatically created for each SNP in order to test the best model. From the standard outputs, GEGEINTOOL extracts a selected set of parameters (regression coefficients, p-values, adjusted means, etc., and groups them in a single MS Excel Spreadsheet. The tool allows editing the set of filter parameters, filtering the selected results depending on p-values, as well as plotting the selected gene-gene interactions to check consistency. In conclusion, GEGEINTOOL is a useful and friendly tool for exploring and identifying gene-gene interactions in complex diseases.

  13. Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers

    Directory of Open Access Journals (Sweden)

    Durnford Dion G

    2008-02-01

    Full Text Available Abstract Background The sulfate assimilation pathway is present in photosynthetic organisms, fungi, and many bacteria, providing reduced sulfur for the synthesis of cysteine and methionine and a range of other metabolites. In photosynthetic eukaryotes sulfate is reduced in the plastids whereas in aplastidic eukaryotes the pathway is cytosolic. The only known exception is Euglena gracilis, where the pathway is localized in mitochondria. To obtain an insight into the evolution of the sulfate assimilation pathway in eukaryotes and relationships of the differently compartmentalized isoforms we determined the locations of the pathway in lineages for which this was unknown and performed detailed phylogenetic analyses of three enzymes involved in sulfate reduction: ATP sulfurylase (ATPS, adenosine 5'-phosphosulfate reductase (APR and sulfite reductase (SiR. Results The inheritance of ATPS, APR and the related 3'-phosphoadenosine 5'-phosphosulfate reductase (PAPR are remarkable, with multiple origins in the lineages that comprise the opisthokonts, different isoforms in chlorophytes and streptophytes, gene fusions with other enzymes of the pathway, evidence a eukaryote to prokaryote lateral gene transfer, changes in substrate specificity and two reversals of cellular location of host- and endosymbiont-originating enzymes. We also found that the ATPS and APR active in the mitochondria of Euglena were inherited from its secondary, green algal plastid. Conclusion Our results reveal a complex history for the enzymes of the sulfate assimilation pathway. Whilst they shed light on the origin of some characterised novelties, such as a recently described novel isoform of APR from Bryophytes and the origin of the pathway active in the mitochondria of Euglenids, the many distinct and novel isoforms identified here represent an excellent resource for detailed biochemical studies of the enzyme structure/function relationships.

  14. miRSel: Automated extraction of associations between microRNAs and genes from the biomedical literature

    Directory of Open Access Journals (Sweden)

    Zimmer Ralf

    2010-03-01

    Full Text Available Abstract Background MicroRNAs have been discovered as important regulators of gene expression. To identify the target genes of microRNAs, several databases and prediction algorithms have been developed. Only few experimentally confirmed microRNA targets are available in databases. Many of the microRNA targets stored in databases were derived from large-scale experiments that are considered not very reliable. We propose to use text mining of publication abstracts for extracting microRNA-gene associations including microRNA-target relations to complement current repositories. Results The microRNA-gene association database miRSel combines text-mining results with existing databases and computational predictions. Text mining enables the reliable extraction of microRNA, gene and protein occurrences as well as their relationships from texts. Thereby, we increased the number of human, mouse and rat miRNA-gene associations by at least three-fold as compared to e.g. TarBase, a resource for miRNA-gene associations. Conclusions Our database miRSel offers the currently largest collection of literature derived miRNA-gene associations. Comprehensive collections of miRNA-gene associations are important for the development of miRNA target prediction tools and the analysis of regulatory networks. miRSel is updated daily and can be queried using a web-based interface via microRNA identifiers, gene and protein names, PubMed queries as well as gene ontology (GO terms. miRSel is freely available online at http://services.bio.ifi.lmu.de/mirsel.

  15. The eukaryotic fossil record in deep time

    Science.gov (United States)

    Butterfield, N.

    2011-12-01

    Eukaryotic organisms are defining constituents of the Phanerozoic biosphere, but they also extend well back into the Proterozoic record, primarily in the form of microscopic body fossils. Criteria for identifying pre-Ediacaran eukaryotes include large cell size, morphologically complex cell walls and/or the recognition of diagnostically eukaryotic cell division patterns. The oldest unambiguous eukaryote currently on record is an acanthomorphic acritarch (Tappania) from the Palaeoproterozoic Semri Group of central India. Older candidate eukaryotes are difficult to distinguish from giant bacteria, prokaryotic colonies or diagenetic artefacts. In younger Meso- and Neoproterozoic strata, the challenge is to recognize particular grades and clades of eukaryotes, and to document their macro-evolutionary expression. Distinctive unicellular forms include mid-Neoproterozoic testate amoebae and phosphate biomineralizing 'scale-microfossils' comparable to an extant green alga. There is also a significant record of seaweeds, possible fungi and problematica from this interval, documenting multiple independent experiments in eukaryotic multicellularity. Taxonomically resolved forms include a bangiacean red alga and probable vaucheriacean chromalveolate algae from the late Mesoproterozoic, and populations of hydrodictyacean and siphonocladalean green algae of mid Neoproterozoic age. Despite this phylogenetic breadth, however, or arguments from molecular clocks, there is no convincing evidence for pre-Ediacaran metazoans or metaphytes. The conspicuously incomplete nature of the Proterozoic record makes it difficult to resolve larger-scale ecological and evolutionary patterns. Even so, both body fossils and biomarker data point to a pre-Ediacaran biosphere dominated overwhelming by prokaryotes. Contemporaneous eukaryotes appear to be limited to conspicuously shallow water environments, and exhibit fundamentally lower levels of morphological diversity and evolutionary turnover than

  16. The origin and diversification of eukaryotes: problems with molecular phylogenetics and molecular clock estimation.

    Science.gov (United States)

    Roger, Andrew J; Hug, Laura A

    2006-06-29

    Determining the relationships among and divergence times for the major eukaryotic lineages remains one of the most important and controversial outstanding problems in evolutionary biology. The sequencing and phylogenetic analyses of ribosomal RNA (rRNA) genes led to the first nearly comprehensive phylogenies of eukaryotes in the late 1980s, and supported a view where cellular complexity was acquired during the divergence of extant unicellular eukaryote lineages. More recently, however, refinements in analytical methods coupled with the availability of many additional genes for phylogenetic analysis showed that much of the deep structure of early rRNA trees was artefactual. Recent phylogenetic analyses of a multiple genes and the discovery of important molecular and ultrastructural phylogenetic characters have resolved eukaryotic diversity into six major hypothetical groups. Yet relationships among these groups remain poorly understood because of saturation of sequence changes on the billion-year time-scale, possible rapid radiations of major lineages, phylogenetic artefacts and endosymbiotic or lateral gene transfer among eukaryotes. Estimating the divergence dates between the major eukaryote lineages using molecular analyses is even more difficult than phylogenetic estimation. Error in such analyses comes from a myriad of sources including: (i) calibration fossil dates, (ii) the assumed phylogenetic tree, (iii) the nucleotide or amino acid substitution model, (iv) substitution number (branch length) estimates, (v) the model of how rates of evolution change over the tree, (vi) error inherent in the time estimates for a given model and (vii) how multiple gene data are treated. By reanalysing datasets from recently published molecular clock studies, we show that when errors from these various sources are properly accounted for, the confidence intervals on inferred dates can be very large. Furthermore, estimated dates of divergence vary hugely depending on the methods

  17. Complementing the Eukaryotic Protein Interactome.

    Directory of Open Access Journals (Sweden)

    Robert Pesch

    Full Text Available Protein interaction networks are important for the understanding of regulatory mechanisms, for the explanation of experimental data and for the prediction of protein functions. Unfortunately, most interaction data is available only for model organisms. As a possible remedy, the transfer of interactions to organisms of interest is common practice, but it is not clear when interactions can be transferred from one organism to another and, thus, the confidence in the derived interactions is low. Here, we propose to use a rich set of features to train Random Forests in order to score transferred interactions. We evaluated the transfer from a range of eukaryotic organisms to S. cerevisiae using orthologs. Directly transferred interactions to S. cerevisiae are on average only 24% consistent with the current S. cerevisiae interaction network. By using commonly applied filter approaches the transfer precision can be improved, but at the cost of a large decrease in the number of transferred interactions. Our Random Forest approach uses various features derived from both the target and the source network as well as the ortholog annotations to assign confidence values to transferred interactions. Thereby, we could increase the average transfer consistency to 85%, while still transferring almost 70% of all correctly transferable interactions. We tested our approach for the transfer of interactions to other species and showed that our approach outperforms competing methods for the transfer of interactions to species where no experimental knowledge is available. Finally, we applied our predictor to score transferred interactions to 83 targets species and we were able to extend the available interactome of B. taurus, M. musculus and G. gallus with over 40,000 interactions each. Our transferred interaction networks are publicly available via our web interface, which allows to inspect and download transferred interaction sets of different sizes, for various

  18. Microbial eukaryotic distributions and diversity patterns in a deep-sea methane seep ecosystem.

    Science.gov (United States)

    Pasulka, Alexis L; Levin, Lisa A; Steele, Josh A; Case, David H; Landry, Michael R; Orphan, Victoria J

    2016-09-01

    Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function.

  19. Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers

    Indian Academy of Sciences (India)

    Prashant K Pandey; Jay Siddharth; Pankaj Verma; Ashish Bavdekar; Milind S Patole; Yogesh S Shouche

    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 micro-eukaryotes 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.

  20. Crystal structure of the eukaryotic ribosome.

    Science.gov (United States)

    Ben-Shem, Adam; Jenner, Lasse; Yusupova, Gulnara; Yusupov, Marat

    2010-11-26

    Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.

  1. Microsatellite Marker Content Mapping of 12 Candidate Genes for Obesity: Assembly of Seven Obesity Screening Panels for Automated Genotyping

    OpenAIRE

    Winick, Jeffrey D.; Friedman, Jeffrey M.

    1998-01-01

    Twin studies, adoption studies, and studies of familial aggregation indicate that obesity has a genetic component. Whereas, the genetic factors predisposing to obesity have been elucidated for several rare syndromes, the factors responsible for obesity in the general population have remained elusive. Genetic studies of complex traits are often accelerated by the use of candidate genes. To facilitate genetic studies of human obesity, seven multiplex panels of candidate genes for obesity that a...

  2. 大鼠Kir6.2基因真核表达载体的构建及其在HEK293细胞中的表达%Construction of a eukaryotic expression vector containing the Kir6.2 gene and its expression in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    任晓燕; 谭淑慧; 马国诏; 杜怡峰

    2011-01-01

    Objective To clone the Kir6.2 gene, construct its eukaryotic expression vector, and obtain positive HEK293 cell clones stably expressing the Kir6.2 gene.Methods Total RNA was extracted from the SD rat brain.Full-length cDNA encoding the Kir6.2 gene was obtained by RT-PCR and inserted into the T1-simple cloning vector.After the sequencing was confirmed , the gene was subcloned into pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3.0/Kir6.2.The recombinant plasmid was transfected into HEK293 cells by lipofectamin and positive cell clones were screened with G418.Expression of the Kir6.2 gene in the transfected cells was confirmed by RT-PCR and Western blot.Results PCR and sequencing showed that the target gene was cloned into the recombinant vector.Overexpression of the Kir6.2 gene in the transfected HEK293 cells was identified by RT-PCR and Western blot.Conclusion The eukaryotic expression plasmid containing the Kir6.2 gene was successfully constructed.Positive HEK293 cell clones stably expressing the Kir6.2 gene were obtained.%目的 克隆Kit6.2基因,构建真核表达载体pcDNA3.0/Kir6.2,将重组质粒转染HEK293细胞获得高表达Kir6.2基因的细胞克隆.方法 从SD大鼠脑组织中提取总RNA,采用RT-PCR方法获得Kit6.2基因的全长cDNA,插入TI-simple克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.0,利用脂质体将重组质粒转染HEK293细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR和Western blot方法,鉴定Kir6.2基因在HEK293细胞中的过表达.结果 经PCR和DNA序列测定证实,目的 基因已插入重组质粒,RT-PCR和West-em blot证明,经G418筛选得到的转基因HEK293细胞克隆中存在Kir6.2基因的表达.结论 成功构建Kir6.2基因的真核表达载体,获得稳定表达Kil6.2基因的HEK293 细胞克隆.

  3. Phylogenomic study indicates widespread lateral gene transfer in Entamoeba and suggests a past intimate relationship with parabasalids.

    Science.gov (United States)

    Grant, Jessica R; Katz, Laura A

    2014-09-01

    Lateral gene transfer (LGT) has impacted the evolutionary history of eukaryotes, though to a lesser extent than in bacteria and archaea. Detecting LGT and distinguishing it from single gene tree artifacts is difficult, particularly when considering very ancient events (i.e., over hundreds of millions of years). Here, we use two independent lines of evidence--a taxon-rich phylogenetic approach and an assessment of the patterns of gene presence/absence--to evaluate the extent of LGT in the parasitic amoebozoan genus Entamoeba. Previous work has suggested that a number of genes in the genome of Entamoeba spp. were acquired by LGT. Our approach, using an automated phylogenomic pipeline to build taxon-rich gene trees, suggests that LGT is more extensive than previously thought. Our analyses reveal that genes have frequently entered the Entamoeba genome via nonvertical events, including at least 116 genes acquired directly from bacteria or archaea, plus an additional 22 genes in which Entamoeba plus one other eukaryote are nested among bacteria and/or archaea. These genes may make good candidates for novel therapeutics, as drugs targeting these genes are less likely to impact the human host. Although we recognize the challenges of inferring intradomain transfers given systematic errors in gene trees, we find 109 genes supporting LGT from a eukaryote to Entamoeba spp., and 178 genes unique to Entamoeba spp. and one other eukaryotic taxon (i.e., presence/absence data). Inspection of these intradomain LGTs provide evidence of a common sister relationship between genes of Entamoeba (Amoebozoa) and parabasalids (Excavata). We speculate that this indicates a past close relationship (e.g., symbiosis) between ancestors of these extant lineages.

  4. Sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus.

    Science.gov (United States)

    Bell, Philip John Livingstone

    2006-11-07

    The origin of the eukaryotic cell cycle, including mitosis, meiosis, and sex are as yet unresolved aspects of the evolution of the eukaryotes. The wide phylogenetic distribution of both mitosis and meiosis suggest that these processes are integrally related to the origin of the earliest eukaryotic cells. According to the viral eukaryogenesis (VE) hypothesis, the eukaryotes are a composite of three phylogenetically unrelated organisms: a viral lysogen that evolved into the nucleus, an archaeal cell that evolved into the eukaryotic cytoplasm, and an alpha-proteobacterium that evolved into the mitochondria. In the extended VE hypothesis presented here, the eukaryotic cell cycle arises as a consequence of the derivation of the nucleus from a lysogenic DNA virus.

  5. Nucleoporin Nup98: a gatekeeper in the eukaryotic kingdoms.

    Science.gov (United States)

    Iwamoto, Masaaki; Asakawa, Haruhiko; Hiraoka, Yasushi; Haraguchi, Tokuko

    2010-06-01

    The nucleoporin Nup98 is an essential component of the nuclear pore complex. This peripheral nucleoporin with its Gly-Leu-Phe-Gly (GLFG) repeat domain contributes to nuclear-cytoplasmic trafficking, including mRNA export. In addition, accumulating studies indicate that Nup98 plays roles in several important biological events such as gene expression, mitotic checkpoint, and pathogenesis. Nup98 is well conserved among organisms belonging to the fungi and animal kingdoms. These kingdoms belong to the eukaryotic supergroup Opisthokonta. However, there is considerable diversity in the Nup98 orthologs expressed in organisms belonging to other eukaryotic supergroups. Intriguingly, in ciliates, a unicellular organism having two functionally distinct nuclei, GLFG-Nup98 is present in one of the nuclei and a distinct Nup98 ortholog is present in the other nucleus, and these different Nup98s participate in a nucleus-selective transport mechanism. In this review, we focus on Nup98 function and discuss how this nucleoporin has evolved in eukaryotic kingdoms.

  6. Expression ofpprI andpprA genes from deinococcus radiodurans in eukaryotic 293T cells%耐辐射奇球菌pprI和pprA基因在真核细胞293T中的表达

    Institute of Scientific and Technical Information of China (English)

    肖潇; 马云; 肖方竹; 唐艳; 杨奇; 黄波; 唐旻; 何淑雅

    2016-01-01

    To construct fluorescence expression plasmids pEGFP-N1-pprA and pDsRed1-N1-flag-pprI, pGADT-7-pprA and pet28a-pprI plasmid constructed at an earlier laboratory stage was used as the template, and Lipofectamine 2000 was employed to transfect two recombinant vectors into 293T cells. A fluorescent microscope was used for Fluorescence observation, and Western blot was employed to examine the expression of the fusion protein. Double digestions and the agarose gel electrophoresis showed that target bands appeared at 4700 bp and 1000 bp, 4800 bp and 1100 bp. No apparent frameshift mutation occurred as shown in the sequencing results. The fluorescent microscopy showed red and green phosphors; the Western blot results indicated protein expression at 65 kDa and 60 kDa. pDsRed1-N1-flag-pprI and pEGFP-N1-pprA were successfully constructed to express proteins for eukaryotic cells 293T in vitro. The results indicated that prokaryotic genespprA and pprI could co-express proteins in eukaryotic cells successfully, and laid a foundation for the interaction and synergism ofpprA,pprI, and their products in the regulation network of radiation verified by DR, enhancing the radiation resistance of eukaryotic cells.%以pGADT-7-pprA、pet28a-pprI载体为模板,构建pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体,脂质体2000介导将两个重组载体共转入293T细胞.双酶切及琼脂糖凝胶电泳显示在4700、1000 bp处与4800、1100 bp处出现目的条带.测序结果显示构建序列与模板序列一致,氨基酸序列100%正确.荧光显微镜下见到红色和绿色荧光;Western blot结果显示在不同检测水平65 kDa及60 kDa大小处有融合蛋白表达.结果提示pEGFP-N1-pprA、pDsRed1-N1-flag-pprI真核表达载体构建成功,并在离体293T细胞中共同表达蛋白.证明原核基因pprI、pprA能够在真核细胞中共表达,为后续实验验证DR菌高抗性基因pprA、pprI及其产物在辐射调控网络中的相互作用和协同作用、

  7. A Droplet Microfluidic Platform for Automating Genetic Engineering.

    Science.gov (United States)

    Gach, Philip C; Shih, Steve C C; Sustarich, Jess; Keasling, Jay D; Hillson, Nathan J; Adams, Paul D; Singh, Anup K

    2016-05-20

    We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

  8. Gene: a gene-centered information resource at NCBI.

    Science.gov (United States)

    Brown, Garth R; Hem, Vichet; Katz, Kenneth S; Ovetsky, Michael; Wallin, Craig; Ermolaeva, Olga; Tolstoy, Igor; Tatusova, Tatiana; Pruitt, Kim D; Maglott, Donna R; Murphy, Terence D

    2015-01-01

    The National Center for Biotechnology Information's (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) integrates gene-specific information from multiple data sources. NCBI Reference Sequence (RefSeq) genomes for viruses, prokaryotes and eukaryotes are the primary foundation for Gene records in that they form the critical association between sequence and a tracked gene upon which additional functional and descriptive content is anchored. Additional content is integrated based on the genomic location and RefSeq transcript and protein sequence data. The content of a Gene record represents the integration of curation and automated processing from RefSeq, collaborating model organism databases, consortia such as Gene Ontology, and other databases within NCBI. Records in Gene are assigned unique, tracked integers as identifiers. The content (citations, nomenclature, genomic location, gene products and their attributes, phenotypes, sequences, interactions, variation details, maps, expression, homologs, protein domains and external databases) is available via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programming utilities (E-Utilities and Entrez Direct) and for bulk transfer by FTP.

  9. Telomeric position effect--a third silencing mechanism in eukaryotes.

    Directory of Open Access Journals (Sweden)

    J Greg Doheny

    Full Text Available Eukaryotic chromosomes terminate in telomeres, complex nucleoprotein structures that are required for chromosome integrity that are implicated in cellular senescence and cancer. The chromatin at the telomere is unique with characteristics of both heterochromatin and euchromatin. The end of the chromosome is capped by a structure that protects the end and is required for maintaining proper chromosome length. Immediately proximal to the cap are the telomere associated satellite-like (TAS sequences. Genes inserted into the TAS sequences are silenced indicating the chromatin environment is incompatible with transcription. This silencing phenomenon is called telomeric position effect (TPE. Two other silencing mechanisms have been identified in eukaryotes, suppressors position effect variegation [Su(vars, greater than 30 members] and Polycomb group proteins (PcG, approximately 15 members. We tested a large number of each group for their ability to suppress TPE [Su(TPE]. Our results showed that only three Su(vars and only one PcG member are involved in TPE, suggesting silencing in the TAS sequences occurs via a novel silencing mechanism. Since, prior to this study, only five genes have been identified that are Su(TPEs, we conducted a candidate screen for Su(TPE in Drosophila by testing point mutations in, and deficiencies for, proteins involved in chromatin metabolism. Screening with point mutations identified seven new Su(TPEs and the deficiencies identified 19 regions of the Drosophila genome that harbor suppressor mutations. Chromatin immunoprecipitation experiments on a subset of the new Su(TPEs confirm they act directly on the gene inserted into the telomere. Since the Su(TPEs do not overlap significantly with either PcGs or Su(vars, and the candidates were selected because they are involved generally in chromatin metabolism and act at a wide variety of sites within the genome, we propose that the Su(TPE represent a third, widely used, silencing

  10. Challenges in Whole-Genome Annotation of Pyrosequenced Eukaryotic Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Alan; Grigoriev, Igor

    2009-04-17

    Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentous ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.

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

    Science.gov (United States)

    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

  12. CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters.

    Science.gov (United States)

    Weber, T; Rausch, C; Lopez, P; Hoof, I; Gaykova, V; Huson, D H; Wohlleben, W

    2009-03-10

    Bacterial secondary metabolites are an important source of antimicrobial and cytostatic drugs. These molecules are often synthesized in a stepwise fashion by multimodular megaenzymes that are encoded in clusters of genes encoding enzymes for precursor supply and modification. In this work,we present an open source software pipeline, CLUSEAN (CLUster SEquence ANalyzer) that helps to annotate and analyze such gene clusters. CLUSEAN integrates standard analysis tools, like BLAST and HMMer, with specific tools for the identification of the functional domains and motifs in nonribosomal peptide synthetases (NRPS)/type I polyketide synthases (PKS) and the prediction of specificities of NRPS.

  13. Construction of eukaryotic expression plasmid of gene MIC3 of Toxoplasma Gondii and its expression in BHK cells%弓形虫MIC3基因真核表达质粒的构建及其在BHK细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    唐莉娜; 刘涛; 黄雨婷; 徐丽娜; 卢丽丹

    2012-01-01

    目的 构建弓形虫微线体蛋白( MIC3)的真核表达质粒,为进一步研究MIC3蛋白的功能奠定基础.方法 PCR法扩增弓形虫MIC3目的基因,将纯化的目的基因插入到真核表达质粒PCDNA3.0,并转入DH5α 宿主菌中,在含有青霉素的SOB平板上筛选阳性重组子,采用酶切和PCR扩增法对重组质粒进行鉴定;应用HifectinⅡ真核细胞转染试剂将弓形虫PCDNA-MIC3真核表达质粒转染幼地鼠肾细胞(BHK),表达产物用SDS-PAGE进一步确认,ELISA法检测表达蛋白的免疫原性.结果 重组质粒PCDNA-MIC3经单双酶切及PCR扩增都得到以原插入片段MIC3基因1120 bp大小相同的片段,PCDNA-MIC3能在BHK细胞中高效表达,表达产物能被弓形虫特异性血清所识别.结论 成功构建了弓形虫MIC3基因真核表达质粒PCDNA-MIC3.%Objective To construct eukaryotic expression piasmid of microneme protein MIC3 of Toxoplasma gondii, and so as to lay a foundation for further sturdy on function of protein MIC3. Methods To amplify target gene of Toxoplasma gondii by PCR, insert purified target gene into eukaryotic expression piasmid PCDNA3. 0 and then transfer into host bacteria DH5α, screen positive recombinant on SOB plate that contains penicillin, then carry out identification on the re-combinant plasmid by enzyme digestion and method of PCR amplification, and transfect the constructed eukaryotic expression piasmid of Toxoplasma gondii PCDNA-MIC3 into BHK cells, and further verify the expression product with SDS-PAGE, and test the immunogenicity of expression protein using ELISA, Results For recombinant piasmid PCDNA-MIC3, either by single or double enzyme digestion or PCR amplification, fragment that equals the size of the originally inserted fragment of gene MIC3 1 120 bp were obtained. Toxoplasma gondii PCDNA-MIC3 had high expression in BHK cells, and the expression product could be identified by specific serum of Toxoplasma gondi. Conclusion Eukaryotic expression

  14. Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters

    Science.gov (United States)

    Matsubayashi, Miri; Shimada, Yusuke; Li, Yu-You; Harada, Hideki

    2017-01-01

    Eukaryotic communities in aerobic wastewater treatment processes are well characterized, but little is known about them in anaerobic processes. In this study, abundance, diversity and morphology of eukaryotes in anaerobic sludge digesters were investigated by quantitative real-time PCR (qPCR), 18S rRNA gene clone library construction and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Samples were taken from four different anaerobic sludge digesters in Japan. Results of qPCR of rRNA genes revealed that Eukarya accounted from 0.1% to 1.4% of the total number of microbial rRNA gene copy numbers. The phylogenetic affiliations of a total of 251 clones were Fungi, Alveolata, Viridiplantae, Amoebozoa, Rhizaria, Stramenopiles and Metazoa. Eighty-five percent of the clones showed less than 97.0% sequence identity to described eukaryotes, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown. Clones belonging to the uncultured lineage LKM11 in Cryptomycota of Fungi were most abundant in anaerobic sludge, which accounted for 50% of the total clones. The most dominant OTU in each library belonged to either the LKM11 lineage or the uncultured lineage A31 in Alveolata. Principal coordinate analysis indicated that the eukaryotic and prokaryotic community structures were related. The detection of anaerobic eukaryotes, including the members of the LKM11 and A31 lineages in anaerobic sludge digesters, by CARD-FISH revealed their sizes in the range of 2–8 μm. The diverse and uncultured eukaryotes in the LKM11 and the A31 lineages are common and ecologically relevant members in anaerobic sludge digester. PMID:28264042

  15. Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards.

    Science.gov (United States)

    Williams, L C; Hegde, M R; Herrera, G; Stapleton, P M; Love, D R

    1999-08-01

    Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles.

  16. 喉癌来源的MAGE-A3基因真核表达载体及其稳定表达模型的构建和鉴定%Construction and identification of laryngocarcinoma-derived melanoma antigen-encoding-A3 gene eukaryotic expression vector and steady expression model

    Institute of Scientific and Technical Information of China (English)

    吉晓滨; 吕景礼; 陈敬贤; 刘启才; 谢景华

    2012-01-01

    目的:克隆人黑色素瘤相关抗原MAGE-A3基因,构建真核表达载体,检测、鉴定其在细胞中的表达.方法:MAGE-A3基因进行PCR扩增,凝胶回收PCR产物片段并连接至pMD18-T载体.测序后将MAGE-A3基因片段亚克隆至真核表达载体,构建成pIRES2-EGFP/MAGE-A3重组质粒.经脂质体转染至293T细胞株后,荧光定量PCR、Western-blotting法鉴定MAGE-A3基因的表达.结果:MAGE-A3基因正确克隆在plRES2-EGFP/MAGE-A3,测序结果与GenBank公布的MAGE-A3序列一致.转染293T细胞后能检测到MAGE-A3基因和蛋白的表达.结论:成功构建plRES2-EGFP/MAGE-A3真核表达质粒,并能在293T细胞中有效表达MAGE-A3蛋白,奠定了其作为DNA疫苗应用的基础.%Objective; To construct human melanoma antigen-encoding gene ( MAGE-A3 ) eukaryotic expression vector, and to identify and determine the expression in cell lines. Methods; MAGE-A3 gene was amplified by using polymerase chain reaction (PCR) and was linked to pMD18-T vector after harvesting PCR products via gel extraction. After confirmation by sequencing, the gene segments of MAGE-A3 were subcloned into eukaryotic expression vector for construction of pIRES2-EGFP/MAGE-A3, the recombinant plasmid. This was followed by transfection into the 293 T cell lines using liposomes for further determination of MAGE-A3 gene expression via fluorescent quantitative PCR and Western blotting. Results; MAGE-A3 gene was successfully cloned in the plasmid pIRES2-EGFP/MAGE-A3 , showing identical sequence of MAGE-A3 as compared with that reported in GenBank. The expression of MAGE-A3 genes and proteins were identified after transfection into the 293T cells. Conclusion;The successful construction of eukaryotic expression plasmid pIRES2-EGFP/MAGE-A3 was associated with effective expression of MAGE-A3 protein in 293T cell lines. This may offer grounds for the application of pIRES2-EGFP/MAGE-A3 as a DNA vaccine candidate in laryngocarcinoma immunotherapy.

  17. Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures.

    Science.gov (United States)

    Tojo, Masayoshi; Fujita, Takahiro; Ainoda, Yusuke; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Sakurai, Aki; Masui, Yoshinori; Yazaki, Hirohisa; Takahashi, Hiroshi; Miyoshi-Akiyama, Tohru; Totsuka, Kyoichi; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

  18. Comprehensive comparative analysis of kinesins in photosynthetic eukaryotes

    Directory of Open Access Journals (Sweden)

    Reddy Anireddy SN

    2006-01-01

    Full Text Available Abstract Background Kinesins, a superfamily of molecular motors, use microtubules as tracks and transport diverse cellular cargoes. All kinesins contain a highly conserved ~350 amino acid motor domain. Previous analysis of the completed genome sequence of one flowering plant (Arabidopsis has resulted in identification of 61 kinesins. The recent completion of genome sequencing of several photosynthetic and non-photosynthetic eukaryotes that belong to divergent lineages offers a unique opportunity to conduct a comprehensive comparative analysis of kinesins in plant and non-plant systems and infer their evolutionary relationships. Results We used the kinesin motor domain to identify kinesins in the completed genome sequences of 19 species, including 13 newly sequenced genomes. Among the newly analyzed genomes, six represent photosynthetic eukaryotes. A total of 529 kinesins was used to perform comprehensive analysis of kinesins and to construct gene trees using the Bayesian and parsimony approaches. The previously recognized 14 families of kinesins are resolved as distinct lineages in our inferred gene tree. At least three of the 14 kinesin families are not represented in flowering plants. Chlamydomonas, a green alga that is part of the lineage that includes land plants, has at least nine of the 14 known kinesin families. Seven of ten families present in flowering plants are represented in Chlamydomonas, indicating that these families were retained in both the flowering-plant and green algae lineages. Conclusion The increase in the number of kinesins in flowering plants is due to vast expansion of the Kinesin-14 and Kinesin-7 families. The Kinesin-14 family, which typically contains a C-terminal motor, has many plant kinesins that have the motor domain at the N terminus, in the middle, or the C terminus. Several domains in kinesins are present exclusively either in plant or animal lineages. Addition of novel domains to kinesins in lineage

  19. Alternative Splicing: A Potential Source of Functional Innovation in the Eukaryotic Genome

    OpenAIRE

    Lu Chen; Tovar-Corona, Jaime M.; Urrutia, Araxi O.

    2012-01-01

    Alternative splicing (AS) is a common posttranscriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. The release of the human genome draft revealed a much smaller number of genes than anticipated. Because of its potential role in expanding protein diversity, interest in alternative splicing has been increasing over the last decade. Although recent studies have shown that 94% human multiexon genes undergo AS, evolution of...

  20. Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes.

    Science.gov (United States)

    Oborník, Miroslav; Green, Beverley R

    2005-12-01

    Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.

  1. Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi [corrected]. indicated by metagenomics.

    Science.gov (United States)

    Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

    2014-01-27

    The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Neamphius huxleyi [corrected] . at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Neamphius huxleyi [corrected]. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Neamphius huxleyi [corrected].

  2. Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Lamellomorpha sp. indicated by metagenomics

    Science.gov (United States)

    Li, Zhi-Yong; Wang, Yue-Zhu; He, Li-Ming; Zheng, Hua-Jun

    2014-01-01

    The whole metabolism of a sponge holobiont and the respective contributions of prokaryotic and eukaryotic symbionts and their associations with the sponge host remain largely unclear. Meanwhile, compared with shallow water sponges, deep-sea sponges are rarely understood. Here we report the metagenomic exploration of deep-sea sponge Lamellomorpha sp. at the whole community level. Metagenomic data showed phylogenetically diverse prokaryotes and eukaryotes in Lamellomorpha sp.. MEGAN and gene enrichment analyses indicated different metabolic potentials of prokaryotic symbionts from eukaryotic symbionts, especially in nitrogen and carbon metabolisms, and their molecular interactions with the sponge host. These results supported the hypothesis that prokaryotic and eukaryotic symbionts have different ecological roles and relationships with sponge host. Moreover, vigorous denitrification, and CO2 fixation by chemoautotrophic prokaryotes were suggested for this deep-sea sponge. The study provided novel insights into the respective potentials of prokaryotic and eukaryotic symbionts and their associations with deep-sea sponge Lamellomorpha sp..

  3. Active eukaryotes in microbialites from Highborne Cay, Bahamas, and Hamelin Pool (Shark Bay), Australia.

    Science.gov (United States)

    Edgcomb, Virginia P; Bernhard, Joan M; Summons, Roger E; Orsi, William; Beaudoin, David; Visscher, Pieter T

    2014-02-01

    Microbialites are organosedimentary structures that are formed through the interaction of benthic microbial communities and sediments and include mineral precipitation. These lithifying microbial mat structures include stromatolites and thrombolites. Exuma Sound in the Bahamas, and Hamelin Pool in Shark Bay, Western Australia, are two locations where significant stands of modern microbialites exist. Although prokaryotic diversity in these structures is reasonably well documented, little is known about the eukaryotic component of these communities and their potential to influence sedimentary fabrics through grazing, binding and burrowing activities. Accordingly, comparisons of eukaryotic communities in modern stromatolitic and thrombolitic mats can potentially provide insight into the coexistence of both laminated and clotted mat structures in close proximity to one another. Here we examine this possibility by comparing eukaryotic diversity based on Sanger and high-throughput pyrosequencing of small subunit ribosomal RNA (18S rRNA) genes. Analyses were based on total RNA extracts as template to minimize input from inactive or deceased organisms. Results identified diverse eukaryotic communities particularly stramenopiles, Alveolata, Metazoa, Amoebozoa and Rhizaria within different mat types at both locations, as well as abundant and diverse signatures of eukaryotes with eukaryotic diversity, particularly in hypersaline Hamelin Pool. There was evidence of vertical structuring of protist populations and foraminiferal diversity was highest in bioturbated/clotted thrombolite mats of Highborne Cay.

  4. Automating Finance

    Science.gov (United States)

    Moore, John

    2007-01-01

    In past years, higher education's financial management side has been riddled with manual processes and aging mainframe applications. This article discusses schools which had taken advantage of an array of technologies that automate billing, payment processing, and refund processing in the case of overpayment. The investments are well worth it:…

  5. Automated discovery of tissue-targeting enhancers and transcription factors from binding motif and gene function data.

    Directory of Open Access Journals (Sweden)

    Geetu Tuteja

    2014-01-01

    Full Text Available Identifying enhancers regulating gene expression remains an important and challenging task. While recent sequencing-based methods provide epigenomic characteristics that correlate well with enhancer activity, it remains onerous to comprehensively identify all enhancers across development. Here we introduce a computational framework to identify tissue-specific enhancers evolving under purifying selection. First, we incorporate high-confidence binding site predictions with target gene functional enrichment analysis to identify transcription factors (TFs likely functioning in a particular context. We then search the genome for clusters of binding sites for these TFs, overcoming previous constraints associated with biased manual curation of TFs or enhancers. Applying our method to the placenta, we find 33 known and implicate 17 novel TFs in placental function, and discover 2,216 putative placenta enhancers. Using luciferase reporter assays, 31/36 (86% tested candidates drive activity in placental cells. Our predictions agree well with recent epigenomic data in human and mouse, yet over half our loci, including 7/8 (87% tested regions, are novel. Finally, we establish that our method is generalizable by applying it to 5 additional tissues: heart, pancreas, blood vessel, bone marrow, and liver.

  6. How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

    Directory of Open Access Journals (Sweden)

    Pawlowski Jan

    2004-06-01

    Full Text Available Abstract Background Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES, we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland. Results Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1 the presence of undetected chimeric sequences; (2 the misplacement of several fast-evolving sequences; and (3 the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups. Conclusions Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel

  7. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-I2 and IFN-γ,and may be play an important part in the process of controlling acute phase of toxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH Ⅰ and EcoR Ⅰ restriction enzyme site. The cDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector.After cloned, the vector was digested with BamH Ⅰ and EcoR Ⅰ and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAXI-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasma gondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用.本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHⅠ和EcoRⅠ酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因.PCR产物连接到pMD18-T克隆载体.用限

  8. Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells%刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    李运娜; 黄金贵; 李建华; 宫鹏涛; 杨举; 李赫; 李淑红; 张西臣

    2011-01-01

    Cyclophilins (CyPs) are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-12 and IFN-γ and may be play an important part in the process of controlling acute phase oftoxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH I and EcoR I restriction enzyme site. The eDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector. After cloned, the vector was digested with BamH I and EcoR I and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAX1-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.%亲环蛋白(cyclophilin,CyP)是一类广泛存在于原核和真核生物体内的胞溶性蛋白,是刚地弓形虫(Toxoplasmngondii)速殖子的主要成分,能够诱导产生IL-12和IFN-γ,在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列,设计并合成一对包含BamHI和EcoRI酶切位点的引物,以cDNA为模板,应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载

  9. EuPathDB: the eukaryotic pathogen genomics database resource

    Science.gov (United States)

    Aurrecoechea, Cristina; Barreto, Ana; Basenko, Evelina Y.; Brestelli, John; Brunk, Brian P.; Cade, Shon; Crouch, Kathryn; Doherty, Ryan; Falke, Dave; Fischer, Steve; Gajria, Bindu; Harb, Omar S.; Heiges, Mark; Hertz-Fowler, Christiane; Hu, Sufen; Iodice, John; Kissinger, Jessica C.; Lawrence, Cris; Li, Wei; Pinney, Deborah F.; Pulman, Jane A.; Roos, David S.; Shanmugasundram, Achchuthan; Silva-Franco, Fatima; Steinbiss, Sascha; Stoeckert, Christian J.; Spruill, Drew; Wang, Haiming; Warrenfeltz, Susanne; Zheng, Jie

    2017-01-01

    The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host–pathogen interactions. PMID:27903906

  10. EuPathDB: the eukaryotic pathogen genomics database resource.

    Science.gov (United States)

    Aurrecoechea, Cristina; Barreto, Ana; Basenko, Evelina Y; Brestelli, John; Brunk, Brian P; Cade, Shon; Crouch, Kathryn; Doherty, Ryan; Falke, Dave; Fischer, Steve; Gajria, Bindu; Harb, Omar S; Heiges, Mark; Hertz-Fowler, Christiane; Hu, Sufen; Iodice, John; Kissinger, Jessica C; Lawrence, Cris; Li, Wei; Pinney, Deborah F; Pulman, Jane A; Roos, David S; Shanmugasundram, Achchuthan; Silva-Franco, Fatima; Steinbiss, Sascha; Stoeckert, Christian J; Spruill, Drew; Wang, Haiming; Warrenfeltz, Susanne; Zheng, Jie

    2017-01-04

    The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host-pathogen interactions.

  11. Análisis automático de zooplancton utilizando imágenes digitalizadas: estado del conocimiento y perspectivas en Latinoamérica

    Directory of Open Access Journals (Sweden)

    Johanna Medellín-Mora

    2013-03-01

    Full Text Available Los estudios de comunidades de zooplancton son de gran importancia para la biología, ecología y conservación del ecosistema marino. Sin embargo, estas investigaciones implican un proceso de alto costo en términos de tiempo y esfuerzo, requiriendo personal entrenado para la identificación, conteo y medición de numerosos grupos taxonómicos, así como para la estimación de biomasa. Actualmente, existe un creciente interés por la implementación de nuevas técnicas que permitan automatizar los análisis, incorporando tecnología informática aplicada al examen automático de imágenes digitalizadas. En este trabajo se revisa el estado del conocimiento de algunas metodologías, basadas en el estudio de imágenes digitalizadas de zooplancton. Se presenta una descripción general de los métodos conocidos, tales como ZooScan-ZooProcess y Zoolmage, se analizan sus resultados para la identificación de distintos zooplancteres, se comparan con el método tradicional y se hacen propuestas para mejorar su aplicación. Adicionalmente, se resumen las experiencias con estos sistemas en el análisis de patrones de variabilidad espacial y temporal de la comunidad zooplanctónica, basados en el espectro de tamaño y pruebas para la estimación de biomasa de zooplancton. Finalmente, se elabora un listado de capacidades y limitaciones registradas en la bibliografía reciente, y se discute sobre las perspectivas de desarrollo y aplicación que ofrece esta metodología para la comunidad científica en Latinoamérica.

  12. Construction of recombinant eukaryotic expression plasmid of AMA1 gene of Neospora caninum and its expression in Vero cells%犬新孢子虫AMA1基因重组真核表达质粒的构建及其在Vero细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    郭焕平; 贾立军; 薛书江; 钱年超; 张守发

    2013-01-01

    为构建犬新孢子虫AMA1基因的重组真核表达质粒,了解其在Vero细胞中的表达情况,采用RT-PCR方法扩增牛源犬新孢子虫吉林株AMA1全长基因,构建了重组真核表达质粒pVAX1-NcAMA1;利用脂质体介导转染法将该重组质粒转染至Vero细胞,应用间接免疫荧光方法(IFA)和Western-blot技术检测AMA1基因在Vero细胞中的表达情况.结果显示,扩增的牛源犬新孢子虫吉林株AMA1基因的长度为1 695 bp,与GenBank中相应基因序列(AB265823.1)的同源性为99.9%.成功构建了重组真核表达质粒pVAX1-NcAMA1,IFA检测发现AMA1基因在Vero细胞中获得了瞬时表达,表达产物经Western-blot鉴定,其分子质量约为68 ku,且具有较好的反应原性.本试验为新孢子虫病核酸疫苗的进一步研究奠定了基础.%To construct recombinant eukaryotic expression plasmid of AMA1 gene of Neospora canihum and express it in Vero cells,the full-length fragment of NcAMA1 gene from bovine N.caninum Jilin strain was amplified by RT-PCR,and then subcloned into the expression vector pVAX-1 to construct a eukaryotic expression plasmid pVAX1-AMA1.The recombinant was then transfected into the Vero cells by liposome method.The expressed product in Vero cells was analyzed by indirect immunofluorescence assay (IFA) and Western-blot.The results showed that the amplified fragment was 1 695 bp,and shared 99.9% homology with the corresponding gene sequence in GenBank (AB265823.1).The recombinant eukaryotic expression plasmid pVAX1-AMA1 was successfully constructed.IFA result showed that the AMA1 gene was transiently expressed,and Western-blotting result showed that expressed product was 68 ku in molecular weight and had a positive reaction with anti-AMA1 serum.These results may provide a foundation for further studies on the development of nucleic acid vaccines against N.caninum infections.

  13. 新城疫病毒LaSota株NP、P和L基因表达载体的构建及真核表达%Construction and Eukaryotic Expression of Recombinant Plasmids Containg NP,P and L Genes of NDV La Sota Strain

    Institute of Scientific and Technical Information of China (English)

    呙会会; 刘垒; 周祖涛; 李筱雯; 金卉; 肖运才; 王贵强

    2014-01-01

    To construct the eukaryotic expression vectors containing P,NP and L genes of Newcastle disease virus La Sota strain,explore the mechanism of P,NP and L proteins and the Newcastle disease virus re-verse genetic operating system,according to the sequence of La Sota strain of NDV GenBank (JF950510. 1),4 pairs of specific primers were designed to amplified NP,P and L gene fragments by RT-PCR,and the genes were cloned into the eukaryotic expression vector pCl-neo.By enzyme digestion and sequencing, the recombinant plasmids pCl-NP,pCl-P and pCl-L were transiently transfected into BHK-21 cells.RT-PCR,Western blot and indirect immunofluorescence assay were used to respectively detects the expression of NP,P and L proteins in BHK-21 cells.The recombinant plasmids pCl-NP,pCl-P and pCl-L were con-structed successfully.The results provide a valuable foundation for the construction of Newcastle disease virus reverse genetic operating system.%为研究新城疫病毒NP、P和L蛋白作用机理和新城疫病毒反向遗传操作系统,构建新城疫病毒LaSota株NP、P和L基因的真核表达载体。根据GenBank中新城疫LaSota株的全长序列(JF950510.1),设计4对特异性引物,用RT-PCR法扩增出新城疫病毒LaSota株的NP、P和L基因,将其克隆到真核载体pCl-neo上。通过酶切和测序验证克隆正确,得到重组质粒pCl-NP、pCl-P和pCl-L,瞬时转染到BHK-21细胞中。经RT-PCR、Westernblot、间接免疫荧光试验分别检测NP、P和L蛋白在BHK-21细胞中的表达。经上述三种方法分别检测到NP、P和L蛋白在BHK-21细胞中的表达。重组质粒pCl-NP、pCl-P和pCl-L构建成功,为后期新城疫病毒反向遗传操作系统的构建奠定了基础。

  14. Synthetic biology tools for bioprospecting of natural products in eukaryotes.

    Science.gov (United States)

    Unkles, Shiela E; Valiante, Vito; Mattern, Derek J; Brakhage, Axel A

    2014-04-24

    Filamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast in vivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes.

  15. Enhancer Sharing Promotes Neighborhoods of Transcriptional Regulation Across Eukaryotes

    Science.gov (United States)

    Quintero-Cadena, Porfirio; Sternberg, Paul W.

    2016-01-01

    Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation. PMID:27799341

  16. Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach.

    Science.gov (United States)

    Lentendu, Guillaume; Wubet, Tesfaye; Chatzinotas, Antonis; Wilhelm, Christian; Buscot, François; Schlegel, Martin

    2014-07-01

    To understand the fine-scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long-term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae-Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88,706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic- (farmyard manure), mineral- and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co-occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

  17. Evidence for a Minimal Eukaryotic Phosphoproteome?

    NARCIS (Netherlands)

    Diks, Sander H.; Parikh, Kaushal; van der Sijde, Marijke; Joore, Jos; Ritsema, Tita; Peppelenbosch, Maikel P.

    2007-01-01

    Background. Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes. Methodology/Principal Findings. We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in c

  18. Automated pipeline for atlas-based annotation of gene expresssion patterns: application to postnatal day 7 mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Carson, James P.; Ju, Tao; Bello, Musodiq; Thaller, Christina; Warren, Joe; Kakadiaris, Ioannis; Chiu, Wah; Eichele, Gregor

    2010-02-01

    Abstract As bio-medical images and volumes are being collected at an increasing speed, there is a growing demand for efficient means to organize spatial information for comparative analysis. In many scenarios, such as determining gene expression patterns by in situ hybridization, the images are collected from multiple subjects over a common anatomical region, such as the brain. A fundamental challenge in comparing spatial data from different images is how to account for the shape variations among subjects, which makes direct image-to-image comparison meaningless. In this paper, we describe subdivision meshes as a geometric means to efficiently organize 2D images and 3D volumes collected from different subjects for comparison. The key advantages of a subdivision mesh for this purpose are its light-weight geometric structure and its explicit modeling of anatomical boundaries, which enable efficient and accurate registration. The multi-resolution structure of a subdivision mesh also allows development of fast comparison algorithms among registered images and volumes.

  19. Heating automation

    OpenAIRE

    Tomažič, Tomaž

    2013-01-01

    This degree paper presents usage and operation of peripheral devices with microcontroller for heating automation. The main goal is to make a quality system control for heating three house floors and with that, increase efficiency of heating devices and lower heating expenses. Heat pump, furnace, boiler pump, two floor-heating pumps and two radiator pumps need to be controlled by this system. For work, we have chosen a development kit stm32f4 - discovery with five temperature sensors, LCD disp...

  20. Automation Security

    OpenAIRE

    Mirzoev, Dr. Timur

    2014-01-01

    Web-based Automated Process Control systems are a new type of applications that use the Internet to control industrial processes with the access to the real-time data. Supervisory control and data acquisition (SCADA) networks contain computers and applications that perform key functions in providing essential services and commodities (e.g., electricity, natural gas, gasoline, water, waste treatment, transportation) to all Americans. As such, they are part of the nation s critical infrastructu...

  1. Marketing automation

    OpenAIRE

    Raluca Dania TODOR

    2017-01-01

    The automation of the marketing process seems to be nowadays, the only solution to face the major changes brought by the fast evolution of technology and the continuous increase in supply and demand. In order to achieve the desired marketing results, businessis have to employ digital marketing and communication services. These services are efficient and measurable thanks to the marketing technology used to track, score and implement each campaign. Due to the...

  2. A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

    DEFF Research Database (Denmark)

    Lyngsø, C.; Kjaerulff, S.; Muller, S.;

    2010-01-01

    in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality...

  3. Production of Candida antarctica lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in an ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production.

    Science.gov (United States)

    Hughes, Stephen R; Moser, Bryan R; Harmsen, Amanda J; Bischoff, Kenneth M; Jones, Marjorie A; Pinkelman, Rebecca; Bang, Sookie S; Tasaki, Ken; Doll, Kenneth M; Qureshi, Nasib; Saha, Badal C; Liu, Siqing; Jackson, John S; Robinson, Samantha; Cotta, Michael C; Rich, Joseph O; Caimi, Paolo

    2011-02-01

    A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel.

  4. 碱基切除修复基因HOGG1特异性锤头状核酶表达载体的构建及其功能的初步研究%Constructing the Eukaryotic Expression Vector to Study Preliminarily the Functions of Hammerhead Ribozyme Targeting Base Excision Repair Gene HOGG1

    Institute of Scientific and Technical Information of China (English)

    张遵真; 张勤; 吴媚

    2006-01-01

    Objective Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer A549 cells would be investigated. Methods According to computer design, two specific restriction site BamHⅠ and EcoRⅠ were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by ampicillin resistance, and plasmids were extracted from the positive recombinants and digested by BamH Ⅰ and EcoR Ⅰ, and then were analyzed by agarose gel electrophoresis and DNA sequencing. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. Results The recombinants containing the ribozyme gene

  5. Construction of eukaryotic expression vector and expression of stage-specific gene T314 from newborn larvae of Trichinella spiralis%旋毛虫新生幼虫期特异性T314基因真核表达质粒的构建及表达

    Institute of Scientific and Technical Information of China (English)

    于建立; 白雪; 吴秀萍; 刘明远

    2012-01-01

    目的 构建旋毛虫(Trichinella spirais)新生幼虫期特异性T314基因真核表达质粒,并在BHK细胞中进行表达.方法 从旋毛虫新生幼虫RNA中通过RT-PCR技术扩增无信号肽T314全长基因,定向克隆至真核表达载体pEGFP-N1中,构建重组真核表达质粒T314-pEGFP-N1,脂质体法转染BHK细胞,荧光显微镜观察EGFP的表达,Western blot检测T314融合蛋白的表达.结果 重组真核表达质粒T314-pEGFP-N1经双酶切及测序证实构建正确;转染的BHK细胞48 h转染效率最高;表达的T314融合蛋白可与旋毛虫感染的猪血清发生特异性反应.结论 已成功构建了T314基因重组真核表达质粒T314-pEGFP-N1,并在BHK细胞中融合表达,为进一步研究旋毛虫包囊形成机制奠定了基础.%Objective To construct a eukaryotic expression vector for stage-specific gene T314 from newborn larvae of Trichinella spiralis and express in BHK cells. Methods Signal peptide-free full-length T314 gene was amplified from RNA of newborn larvae of T. Spiralis by RT-PCR and cloned into eukaryotic expression vector Pegfp-Nl. BHK cells were transfected with the constructed recombinant plasmid T314-Pegfp-Nl in mediation of Hposome, then observed for expression of EGFP by fluorescent microscopy, and for that of T314 fusion protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid T314-Pegfp-Nl was constructed correctly. The transfection efficacy of BHK cells reached the maximum 48 h after transfection. The expressed T314 fusion protein showed specific reaction with porcine serum infected with T. Spiralis. Conclusion The recombinant eukaryotic expression vector T314-Pegfp-Nl for T314 gene was successfully constructed, and fusion protein was expressed in BHK cells, which laid a foundation of further study on mechanism of nurse cell formation of T. Spiralis.

  6. Construction of eukaryotic expression plasmid of Fasciola hepatica CatL gene and biological activity analysis of recombinant protein%肝片吸虫组织蛋白酶L真核表达载体构建及重组蛋白活性分析

    Institute of Scientific and Technical Information of China (English)

    闻晓波; 冉旭华; 王春仁; 宋佰芬; 魏晓曼; 李晓娟; 王密; 苗艳

    2013-01-01

    In this research we constructed eukaryotic expression plasmid expressing Fasciola hepalica cathepsin L-like proteases (CatL) and analyzed the immunogenicity of recombinant protein. CatL gene was amplified by PCR with the template of recombinant pET30a-FhCatL plasmid and cloned into pEGFP-Nl vector to construct recombinant plasmids pEGFP-Nl-CatL. The recombinant plasmid pEGFP-Nl-CatL was transfected into HeLa cells and fluorescent signal was detected by fluorescence microscope. The immunogenicity of recombinant protein identified by western blotting demonstrated that the recombinant protein specifically reacted with serum from goat infected by Fasciola hepalica and showed robust immunoreactivity. The eukaryotic expression plasmid may be a potential gene vaccine or diagnose preparations in further study.%目的 构建肝片吸虫组织蛋白酶L(CatL)的真核表达载体,研究重组蛋白的生物学活性.方法 以构建好的重组质粒pET30a-FhCatL为模板,利用PCR技术扩增肝片吸虫组织蛋白酶L基因(catL),连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-N1-CatL,转染HeLa细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况.结果 重组质粒pEGFP-N1-CatL在HeLa细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应.结论 肝片吸虫CatL真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有免疫反应性,可做为分子疫苗的候选和诊断抗原进行进一步的研究.

  7. 猪Mx1基因真核表达载体的构建、鉴定及其表达%Construction,Identification and Expression of Eukaryotic Expression Vector of Pig Mx1 Gene

    Institute of Scientific and Technical Information of China (English)

    覃兆鲜; 潘天彪; 黄光云; 谢炳坤

    2011-01-01

    The purpose of this study was to obtain pig fibroblast cells that transferred Mx1 gene.Total RNA was extracted from pig fibroblast cells that induced with IFN and the eDNA encoding pig Mx1 protein was cloned using reverse transcription polymerase chain reaction (RT-PCR).The Mx1 gene was subcloned into pMSCV-IRES-GFP vector and then the recombinant plasmids were transferred into Luchuan pig fibroblast cells by the method of Lipofectaming 2000.The results showed that Mx1 gene had been integrated into the genome of Luchuan pig fibroblast cells based on the analysis of fluoroscopy and PCR identification.%为获得转Mx1基因的阳性陆川猪成纤维细胞,本研究以干扰素诱导猪成纤维细胞Mx1基因表达,提取细胞总RNA,RT-PCR获得编码猪Mx1蛋白的cDNA;以pMSCV-IRES-GFP为骨架构建猪Mx1基因表达载体pMSCV-IRES-GFP-Mx1,并利用脂质体2000介导重组质粒转染陆川猪胎儿成纤维细胞,通过荧光观察和PCR检测分析结果表明Mx1蛋白基因整合进入陆川猪胎儿成纤维细胞.

  8. Engineering key components in a synthetic eukaryotic signal transduction pathway.

    Science.gov (United States)

    Antunes, Mauricio S; Morey, Kevin J; Tewari-Singh, Neera; Bowen, Tessa A; Smith, J Jeff; Webb, Colleen T; Hellinga, Homme W; Medford, June I

    2009-01-01

    Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB-VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB-VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved-signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.

  9. Construction of Tetracycline-inducible MC4R Gene Eukaryotic Expression Vector in Pig%四环素诱导的猪黑素皮质激素受体-4基因真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    解宇; 汪亮; 张冬杰; 刘娣; 孙亚蒙

    2013-01-01

    Pig melanocortin-4 receptor (MC4R) gene was one of the major genes affect swine growing and finishing, to further study the biological function of the MCAR gene, preparation of high survival rate of transgenic pigs,through the use of Tet gene expression system,promoter regulatory region and transcription element of Tet-on and sequence of pig MCAR gene was amplified by PCR method. The above-mentioned components were linked into pcDNA3. 1( + ) to construct recombinant vector pcDNA3. 1( + )-rtTA-PTight-MC4R by double digestions and connections methods. Restricted enzymes analysis and DNA sequencing showed that the sequence of the recombinant plasmid was correct and fragment was properly connected. Successfully constructed the Tetracycline-inducible pig MC4R eukaryotic expression vector, and in time-specific regulation of expression of the MCAR gene.%猪黑素皮质激素受体-4(melanoeortin-4 receptor,MC4R)基因是影响猪生长肥育的主效基因之一,为了进一步研究MC4R基因的生物学功能,制备高成活率转基因猪,通过采用条件性诱导表达载体系统,PCR方法扩增了猪MC4R基因完整编码区,四环素诱导表达系统(Tet-on)的启动子调控区与转录调节元件.采用双酶切和连接等方法将上述元件连接到pcD-NA3.1(+)上,构建了重组质粒pcDNA3.1(+)-rtTA-PTight-MC4R.经双酶切和测序鉴定,结果显示片段正向连接且片段全长测序正确.试验成功构建了四环素诱导的pcDNA3.1(+)-rtTA-PTight-MC4R真核表达载体,并能在时间特异性方面调控MC4R基因的表达.

  10. 耐辐射球菌pprI基因真核表达载体的构建及其抗辐射作用%Construction of eukaryotic expression vector carrying pprI gene of Deinococcus radiodurans and its radioresistant effect

    Institute of Scientific and Technical Information of China (English)

    文玲; 施怡; 任丽丽; 丛瑛; 杨占山

    2014-01-01

    目的 研究耐辐射球菌pprI原核基因的真核表达载体的构建及其转染对离体真核细胞急性放射损伤的防护作用.方法 应用DNA重组技术构建pEGFP-c1-pprI质粒;采用脂质体转染技术分别将空载体质粒pEGFP-c1和重组质粒pEGFP-c1-pprI转入人肺上皮细胞系Beas-2B细胞,筛选稳定转染细胞系;应用集落形成实验检测受照转染细胞的生存能力;流式细胞仪检测细胞周期和凋亡率的变化;荧光显微镜观察受照细胞ROS荧光强度;免疫荧光实验观察受照细胞不同时间γ-H2AX焦点数目.结果 成功地构建了pprI原核基因的真核表达载体,并在Beas-2B细胞中表达了PprI融合蛋白,建立了稳定转染细胞系.与空白对照组和空载体组相比,细胞克隆生存曲线显示转染了pprI基因的Beas-2B细胞经不同剂量辐射,其存活分数SF增加,该曲线参数D0、Dq、N增高;转染了pprI基因的Beas-2B细胞受照后的ROS的荧光强度和不同时间(1、2、4 h) γ-H2AX的焦点数均显著减低(F=16.73、19.47、6.94,P<0.05);此外,该细胞G2期阻滞(F=139.73、237.92,P<0.05)和凋亡率显著减低(F=626.02、2 052,P<0.05).结论 耐辐射球菌pprI原核基因可在离体真核细胞中稳定表达,并且显著提高受照真核细胞的辐射抗性.%Objective To construct the eukaryotic expression vector of pprI gene from Deinococcus radiodurans R1 and investigate its radioresistant effects in eukaryotic cells.Methods A recombinant vector pEGFP-c1-pprI was constructed by DNA recombinant technique.The empty vector pEGFP-c1 and the pEGFP-c1-pprI were transferred into human lung epithelial cells Beas-2B by LipofectamineTM 2000,respectively.Then the infected cells were screened in order to develop a cell line with stable expression of pprI gene.Cell survival rate was tested by clone-forming assay.Cell cycle distribution and apoptosis were detected by a flow cytometry.The fluorescence intensity of reactive oxygen species (ROS

  11. AMPKα2基因克隆及其野生型和突变型真核表达载体的构建%Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors

    Institute of Scientific and Technical Information of China (English)

    罗招凡; 李芳萍; 丁鹤林; 程桦

    2009-01-01

    背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, AMPK)α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取,其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点.目的:克隆人的AMPKα2基因,并构建其野生型和突变型真核表达载体.设计:单一样本观察.时间及地点:实验于2007-04/2008-01在中山大学附属第二医院临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α为实验室保存.人骨胳肌组织来源于中山大学附属第二医院手术截肢患者,获患者知情同意,新鲜取材后液氮冷冻.方法:采用反转录一聚合酶链反应技术从人骨骼肌扩增AMPKα2基因,并将其克隆到T载体,通过测序对其进行鉴定.采用Quickchange定点诱变试剂盒对AMPKα2基因进行体外定点诱变,并将其野生和突变的编码基因亚克隆到真核表达载体pcDNA3.1中,通过酶切和测序进行鉴定.主要观察指标:①目的基因的克隆.②定点诱变.③真核表达质粒的构建.结果:成功克隆了AMPKα2基因,大小约1 700 bp,与已发表的AMPKα2同源性为99%,GenBank录入号EF056019.成功将AMPKα2第45位Lysine(AAA)突变为Arginine(AGA),成功构建了野生型和突变型pcDNA-AMPK α2重组质粒.结论:实验成功克隆了AMPKα2基因,构建了其野生型和突变型真核表达载体.%BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation

  12. CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID AND SEQUENCE ANALYSIS OF Sj16 GENE OF SCHISTOSOMA JAPONICUM CHINESE STRAIN%日本血吸虫(中国大陆株)Sj16基因真核表达载体的构建及序列分析

    Institute of Scientific and Technical Information of China (English)

    卞国武; 余新炳; 吴忠道; 徐劲; 单志新; 马长玲; 邵筱

    2001-01-01

    目的为了进一步研究日本血吸虫(中国大陆株)Sj16的免疫调节功能,丰富有关血吸虫免疫逃避知识.方法根据曼氏血吸虫Sm1 6基因已知序列设计合成一对引物,用PCR技术从日本血吸虫(中国大陆株)成虫cDNA文库中扩增Sj16基因;将Sj16基因定向克隆入pcDNA3,转化感受态DH5a菌;用酶切、PCR扩增鉴定筛选得到的重组阳性克隆.以重组pcDNA3-Sj16质粒为模板,用双脱氧链末端终止法测定Sj16基因序列,应用软件辅助分析Sj16序列及进行Sj16与曼氏血吸虫的Sm16同源性比较.结果从日本血吸虫(中国大陆株)成虫cDNA文库中获取Sj16基因,重组质粒中含有Sj16基因,成功构建日本血吸虫(中国大陆株)Sj16基因真核表达重组质粒pcDNA3-Sj16;Sj16基因开放读码框有354碱基,编码¨7氨基酸,N端18个氨基酸可能为信号肽序列;Sj16与Sm16有高度同源性,只存在一个氨基酸的差异.结论成功克隆了Sj16基因的真核表达载体,并测定、分析了Sj16基因序列,为深入研究Sj16的免疫调节功能,丰富有关血吸虫免疫逃避知识打下基础.%Objective To further study the immunodulatory function of Schistosoma japonicum Sj16 and more obtain theknowledge about the immune evasion of schistosome. Methods A couple of primers were designed according to theknown sequence of Schistosoma mansoni Sm16 and the Sj16 gene obtained by amplification from cDNA library of S. japon-icum Chinese strain by using PCR technique. By cloning Sj16 gene into a eukaryotic expression vector, pcDNA3, a recombi-nant pcDNA3-Sj16 was constructed and transferred into DH5α. The positive recombinant pcDNA3-Sj16 was screened and i-dentified by agarose gel electrophoresis, endonuclease digestion and PCR technique. The pcDNA3-Sj1 6 recombinant plasmidwas used as template and nucleotide sequence of Sj16 gene was determined by the dideoxy chain termination method. Usingsoftware to analyze the structure and sequence homology of Sj16

  13. Rule-based design of synthetic transcription factors in eukaryotes.

    Science.gov (United States)

    Purcell, Oliver; Peccoud, Jean; Lu, Timothy K

    2014-10-17

    To design and build living systems, synthetic biologists have at their disposal an increasingly large library of naturally derived and synthetic parts. These parts must be combined together in particular orders, orientations, and spacings to achieve desired functionalities. These structural constraints can be viewed as grammatical rules describing how to assemble parts together into larger functional units. Here, we develop a grammar for the design of synthetic transcription factors (sTFs) in eukaryotic cells and implement it within GenoCAD, a Computer-Aided Design (CAD) software for synthetic biology. Knowledge derived from experimental evidence was captured in this grammar to guide the user to create designer transcription factors that should operate as intended. The grammar can be easily updated and refined as our experience with using sTFs in different contexts increases. In combination with grammars that define other synthetic systems, we anticipate that this work will enable the more reliable, efficient, and automated design of synthetic cells with rich functionalities.

  14. Release of chromosomes from the nuclear envelope: a universal mechanism for eukaryotic mitosis?

    Science.gov (United States)

    Kanoh, Junko

    2013-01-01

    Multiple domains of chromosomes are associated with the nuclear envelope (NE) in interphase. The association between chromosomes and the NE is involved in a variety of chromosomal reactions, such as gene expression and DNA repair. However, efficient chromosome movements are required for the fidelity of chromosome segregation in mitosis. Most higher eukaryotes perform open mitosis, in which the NE is broken down, enabling chromosomes to be released from the NE as well as spindle microtubules to access to kinetochores. By contrast, lower eukaryotes, such as Schizosaccharomyces pombe, perform closed mitosis, during which NE breakdown does not occur. In S. pombe, telomeres are tethered to the NE in interphase. Phosphorylation of the telomere-binding protein Rap1 at M phase promotes transient dissociation of telomeres from the NE, facilitating the faithful chromosome segregation. These findings imply a common mechanism for genome stability via the dissociation of chromosomes from the NE in eukaryotic mitosis.

  15. Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.; Bolstad, D; Bolstad, E; Wright, D; Anderson, A

    2009-01-01

    Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

  16. HBD3基因真核表达质粒载体的构建及其体外表达和活性鉴定%Construction and in expression of eukaryotic expression plasmid containing HBD3 gene

    Institute of Scientific and Technical Information of China (English)

    夏章权; 张从纪; 王涛

    2012-01-01

    Objective To further determine the possible effect on antimicrobial activity, plasmid vector containing recombinant human beta defensins 3(HBD3)gene was constructed and the expression of exogenous gene in transformed bone marrow-derived mesenchymal stem cells (BMSCs)was observed. Methods By using RT-PCR obtained HBD3 from gingiva tissue,then recombinat- ed the gene to plasmid Pvivo1-mcs. The recombinated plasmid vector was named as pVIVOl-HBD3 and identified by restriction enzyme analysis and DNA sequencin. After the recombinated plasmid transformed BMSCs, expression of HBD3 in BMSCs was detected by RT-PCR,immunofluorescence and Western blot,and the function was determined by Kleihauer-Betke (K-B) test. Results We successfully constructed recombinant plasmid vector that expressed HBD3. The expression of HBD3 were confirmed by RT-PCR, immunofluorescence and Western blot on transformed BMSCs. The function of HBD3 were confirmed by K-B test. Conclusion The established BMSCs that overexpressed HBD3 provide a new strategy of gene therapy to promote wound healing, especially the infected one.%目的 构建人β防御素3(HBD3)基因真核表达质粒载体,观察其在大鼠骨髓间充质干细胞(BMSCs)中的表达,并检测其活性.方法 利用RT-PCR方法从牙龈组织中扩增出HBD3基因片段,通过DNA重组技术将基因片段重组于pVIVO1-mcs真核表达质粒载体上,构建成pVIVO1-HBD3重组质粒载体,分别用酶切电泳分析及DNA测序的方法对重组质粒载体进行鉴定.运用jetPEI转染试剂将重组质粒转染BMSCs后,RT-PCR、免疫荧光和蛋白质印迹法检测目的 基因的表达.取转染重组质粒的BMSCs无血清上清的浓缩液,采用K-B纸片扩散法进行抗菌活性实验.结果 (1)酶切电泳分析得到相应的目的 片段,大小与理论计算值一致,DNA测序证实目的 基因序列正确无突变,成功构建真核表达质粒载体pVIVO1-HBD3.(2)RT-PCR和免疫荧光、蛋白质印

  17. Gene cloning of human serum albumin and the construction of eukaryotic expression vector%人血清白蛋白基因的克隆及其真核表达载体构建

    Institute of Scientific and Technical Information of China (English)

    洪志勇; 李玉芳; 张兴峰; 林秀娇; 李鸿翔; 庄益芬; 张文昌

    2013-01-01

    利用RT-PCR扩增并克隆了含人血清白蛋白序列长约1.8 kb的片段,酶切鉴定并进行序列分析,测序结果与GenBank中登录的人血清白蛋白cDNA序列的同源性为97%;以鹌鹑卵清蛋白5’调控区POV来启动HAS的表达。用限制性内切酶kpnI和HindⅢ双酶切质粒pOV和pHSA,然后用T4连接酶定向连接成重组质粒pOV-pHSA,经酶切鉴定,两者成功融合。%About 1.8 kb sequence of the fragment of human serum albumin containing were successfully amplified by RT-PCR.The purpose of the initia gene were identified with the help of electrophoresis. The DNA fragment was cloned into vector pGEM-T and transformed into E.coli DH5a host bacteria. Xba Iendonuclease digestion and sequencingfrom selected positive cloned bacteria. The fragment was sequenced,and it was compared with serum albumin cDNA gene of humanin GenBank.The results indicated the homology were 97%.Using 5’-flanking of ovalbumin in quail for the control sequence to activate the expression of HAS.With restrictive interior contact enzyme kpnI with HindⅢ cuts material particle pOV and pHSA,then connects reorganization material particle for pOV-pHSA with the T4 ligase. The fusion vector was sequenced by enzyme digestion.

  18. Automation in biological crystallization.

    Science.gov (United States)

    Stewart, Patrick Shaw; Mueller-Dieckmann, Jochen

    2014-06-01

    Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given.

  19. Automation in biological crystallization

    Science.gov (United States)

    Shaw Stewart, Patrick; Mueller-Dieckmann, Jochen

    2014-01-01

    Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given. PMID:24915074

  20. 真核表达载体pIRES2-EGFP/TRAIL,pIRES2-EGFP/CD的构建和鉴定%Cloning and sequencing of reconstructed eukaryotic expression vector pIRES2-EGFP carrying CD and TRAIL genes

    Institute of Scientific and Technical Information of China (English)

    梁兵; 袁芳; 殷建瑞; 谭丽华; 高庆春; 高聪

    2011-01-01

    Objective To corstruct the eukaryotic expression vector encoding CD and TRAIL genes, pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD, and provide the research basis of the association between overexpression of CD and TRAIL genes in C6 glioma cells.Methods The received plasmids DNA were assessed by electrophoresis in 1% agarose gel and sequencing.CD and TRAIL genes were cloned directionally into eukaryotic expression vector, pIRES2-EGFP, through double enzyme-cutting by Sac Ⅱ/Xho Ⅰ and BamH Ⅰ/Xho Ⅰ.The recombinant plasmids were converted into E.coli DH5α competent cell, and then bolted and identified by double enzyme-cutting of restriction enzyme, PCR, and nucleic acid sequence analysis.Results The length of two pIRES2-EGFP/TRAIL fragments after double-cutting by Sac Ⅱ/Xho Ⅰ were confident with theoretic length of 1.0 kb and 5.2 kb.The length of two pIRES2-EGFP/CD fragments after double-cutting by BamH Ⅰ/Xho Ⅰ were confident with theoretic length of 1.3 kb and 5.2 kb.pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD were confirmed to be contained CD and TRAIL genes by PCR and nucleic acid sequence analysis.Conclusions Reconstructing pIRES2-EGFP/TRAIL and pIRES2-EGFP/CD could successfully establish abasis of further research of the association between overexpression of CD and TRAIL genes in C6 glioma cells.%目的 构建真核表达载体pIRES2-EGFP/TRAIL和pIRES2-EGFP/CD,为研究其联合表达对恶性胶质瘤的联合治疗作用提供基础.方法 将pCMV/CD质粒和pcDNA3.1(+)/TRAIL质粒行琼脂糖凝胶电泳检测,确定其完整性,并进行序列测定确定有无基因突变.pCMV/CD质粒用Sac Ⅱ/ Xho Ⅰ双酶切,pcDNA3.1(+)TRAIL质粒用BamH Ⅰ/XhoⅠ双酶切,将目的 基因定向克隆到真核细胞表达载体pIRES2-EGFP中,转化E.coli DH5α感受态细胞,通过限制性内切酶双酶切、PCR及核酸序列分析等筛选、鉴定重组质粒.结果 所构建的真核表达载体pIRES2-EGFP/TRAIL经Sac Ⅱ/ XhoⅠ双酶切回收片段分别为1

  1. Arsenic and antimony transporters in eukaryotes.

    Science.gov (United States)

    Maciaszczyk-Dziubinska, Ewa; Wawrzycka, Donata; Wysocki, Robert

    2012-01-01

    Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters.

  2. Construction and identification of eukaryotic eukaryotic expression plasmid pcdna3.1-bace and its transient expression in cells

    Institute of Scientific and Technical Information of China (English)

    Huilin Gong; Guanjun Zhang; Weijiang Dong

    2006-01-01

    Objective: To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods: A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy-mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results: The results showed that the cDNAfragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully,and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion: The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the reason of Alzheimer's disease and lays the important foundation for preventing the AD laterly.

  3. Construction of eukaryotic expression plasmid carrying HSV-TK gene and its expression in HepG2 cells%含HSV-TK基因的真核表达载体的构建及其在HepG2细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    张阳德; 孙颖

    2003-01-01

    Objective:To construct a eukaryotic expression plasmid carrying the HSV-TK gene driven by AFP enhancer and CMV promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC).Methods:The minimal essential DNA fragment of AFP gene enhancer was amplified through PCR from genome DNA of HepG2 cells and cloned into the BglII site of plasmid pcDNA3.1-LUC to construct the recombinant plasmid pAFP-LUC. Then the full length cDNA of HSV-TK was cloned into EcoRI site of the recombinant plasmid pAFP-LUC instead of the Luciferase gene to construct pAFP-TK. The recombinant plasmid pAFP-LUC was transferred into AFP-producing hepatoma cell line (HepG2) and non-AFP-producing nonhepatoma cell line (HeLa) by means of lipofectamine. The expression of Luciferase was tested by Luciferase Assay. Results: The length and sequence of AFP enhancer amplified by PCR were confirmed by agarose gel electrophoresis and DNA sequencing.The length, position and orientation of inserted AFP enhancer in pAFP-LUC were all confirmed correct by the methods of restriction digestion and PCR. And it was confirmed by electrophoresis after restriction digestion that the full length HSV-TK was directedly and successfully cloned into the eukaryotic vector . The transcription of Luciferase gene was under the control of AFP enhancer. The expression of Luciferase gene was detected in HepG2 and HeLa cells. The expression of Luciferase is more potent in HepG2 than in HeLa (P<0.05).Conclusions:Construction of a eukaryotic expression plasmid carrying HSV-TK gene driven by AFP enhancer and CMV promotor and its specific expression in HepG2 cells provide a sound basis for targeted gene therapy for HCC.%目的构建AFP增强子CMV启动子调控下的HSV-TK 真核表达质粒用于肝细胞癌的靶向基因治疗.方法采用PCR方法从HepG2细胞基因组DNA中扩增AFP基因增强子最小的功能片断,插入pcDNA3.1-LUC质粒的Bgl II位点,从而构建重组表达质粒pAFP-LUC.HSV-TK cDNA

  4. Construction andimmunogenicity analysis of eukaryotic expression vector of porcine circovirus type 2 Rep gene%猪圆环病毒2型 Rep 基因真核表达载体的构建和免疫原性分析

    Institute of Scientific and Technical Information of China (English)

    王静; 刘成倩; 李红; 易建中; 于宗幸; 陈磊; 孙晓云

    2016-01-01

    根据 GenBank 发表的猪圆环病毒2型(PCV2)基因序列设计并合成1对特异性引物,进行 PCR 扩增,从 PCV2突变株中扩增出 PCV2 Rep 基因,对 PCR 扩增产物清洁后进行双酶切,连接到 pEGFP-C1载体上,转化到大肠杆菌 DH5α感受态细胞中,经 PCR 筛选和测序后鉴定出正确的重组基因,完成真核表达载体的构建。将重组质粒转染到 PK15细胞中,收取病毒液抽提 DNA,PCR 检测到 Rep 基因,证明转染是成功的。进行间接免疫荧光试验,可以检测 Rep 基因在 PK15细胞中的表达,试验结果很好的验证了 Rep 基因的免疫原性。这为进一步研究 PCV2的生物学活性及 PCV2新型疫苗奠定了坚实的基础。%A pair of specific primers was designed and synthesized according to the porcine circovirus type 2(PCV2)gene sequence published in the GenBank,the PCV2 Rep gene was amplified by PCR from mutant strains of PCV2,and after cleaning and double digests the amplified products were connected to the pEGFP-C1 vector and transformed into E.coli DH5αcompetent cells.The correct recombinant gene was identified by PCR screening and sequencing,indicating the construction of eukaryotic expression vector was finished.The recombi-nant plasmid was transfected into PK1 5 cells,and then the virus liquid was collected to extract the DNA.The Rep gene detected by PCR proved that the transfection was successful.The experimental results well verified the im-munogenicity of Rep gene in PK1 5 cells,thus laying a solid foundation for further study of the biological activities and new vaccine of PCV2.

  5. Cloning and Expression of Spider Dragline Silk Protein Gene in Escherichia coli and Eukaryotic Cells%蜘蛛拖牵丝蛋白基因的克隆与表达

    Institute of Scientific and Technical Information of China (English)

    刘丹梅; 王芬; 李文利

    2011-01-01

    蜘蛛丝具有极高的强度和韧度,工业和医学应用价值很高,但由于蜘蛛的不可驯养性使其应用受到限制。因此,本文尝试利用基因工程的方法获得蛛丝蛋白的表达。我们利用巢式PCR技术从大腹圆蛛Araneus ventricosus基因组中克隆了长度为837bp的拖牵丝蛋白基因(ASP),并分别将其构建至原核表达载体pGEX-6p-1和真核表达载体pGFP-N2上,分别命名为pASG和pASN。pASG在大肠杆菌中16℃下24h诱导表达后,经蛋白质印迹证明成功地表达了GST-ASP融合蛋白;pASN转染昆虫sf9细胞48h%The extreme tensile strength and toughness of spider silk make it a superior candidate for a wide range of medical and industrial applications. In this study, a 837 bp fragment which encode dragline silk gene (ASP) was cloned from the genome of spider (Ar

  6. Construction of eukaryotic expression system of human cannabinoid receptor 2 gene and its expression in HEK293 cells%人Ⅱ型大麻受体真核表达体系构建及其在 HEK293细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    孙厚良; 李晶; 龙明; 封玉玲

    2015-01-01

    目的:构建人Ⅱ型大麻受体( hCB2)基因GV230真核表达质粒,并检测hCB2基因在HEK293细胞中的表达。方法利用人脑皮质细胞的总RNA为模板,RT-PCR获得cDNA,通过酶切、连接及测序鉴定正确后,再将目的片段插入真核表达载体GV230,构建重组表达质粒GV230-hCB2,阳性克隆用脂质体瞬时转染HEK293细胞。激光共聚焦扫描显微镜和Western blotting法检测hCB2基因表达产物在细胞的表达情况。结果扩增出hCB2基因片段,成功构建了重组表达质粒,并检测到目的蛋白在转染细胞中表达,观察到hCB2受体在胞膜分布和表达。结论成功构建GV230-hCB2质粒,该质粒在HEK293细胞中能表达hCB2蛋白,此为进一步研究hCB2生物学功能奠定了实验基础。%Objective To construct the eukaryotic expression plasmid of human cannabinoid receptor 2 ( hCB2) gene GV230 and to detect the expression of hCB2 gene in the HEK293 cells.Methods Full length of hCB2 cDNA was obtained by RT-PCR with total RNA isolated from human T lymphocytes, and then we inserted the target fragment into eukaryotic ex-pression vector GV230 to construct the recombinant expression plasmid GV230-hCB2 after enzyme digestion, connection and sequencing.We used the liposome to transiently transfect HEK293 cells.The expression of hCB2 gene expression products was detected by Western blotting and confocal laser scanning microscope (CLSM).Results The hCB2 gene fragments were amplified, and we successfully constructed the recombinant expression plasmid, detected the target protein expressing in the transfected cells, and observed the distribution and expression of hCB2 receptor in the cell membrane. Conclusion We successfully contrast the GV230-hCB2 plasmid expressing hCB2 protein in the HEK293 cells, which lays the experimental foundation for further research of hCB2 biology function.

  7. Automated Budget System

    Data.gov (United States)

    Department of Transportation — The Automated Budget System (ABS) automates management and planning of the Mike Monroney Aeronautical Center (MMAC) budget by providing enhanced capability to plan,...

  8. Alternative Splicing: A Potential Source of Functional Innovation in the Eukaryotic Genome

    Directory of Open Access Journals (Sweden)

    Lu Chen

    2012-01-01

    Full Text Available Alternative splicing (AS is a common posttranscriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. The release of the human genome draft revealed a much smaller number of genes than anticipated. Because of its potential role in expanding protein diversity, interest in alternative splicing has been increasing over the last decade. Although recent studies have shown that 94% human multiexon genes undergo AS, evolution of AS and thus its potential role in functional innovation in eukaryotic genomes remain largely unexplored. Here we review available evidence regarding the evolution of AS prevalence and functional role. In addition we stress the need to correct for the strong effect of transcript coverage in AS detection and set out a strategy to ultimately elucidate the extent of the role of AS in functional innovation on a genomic scale.

  9. 日本血吸虫26kDa谷胱甘肽S-转移酶基因真核表达载体的构建及序列测定%CONSTRUCTION AND SEQUENCING OF 26-KILODALTON GLUTATHIONE TRANSFERASES GENE OF SCHISTOSOMA JAPONICUM INTO EUKARYOTIC EXPRESSION VECTOR

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    目的扩增日本血吸虫26kDa谷胱甘肽S-转移酶(Sj26GST)基因片段,构建其重组真核表达载体,以研制SjGST核酸疫苗。方法根据已知基因序列设计一对引物,运用RT-PCR技术扩增Sj26GST目的基因片段,将其克隆到pcD-NA3质粒中,并经酶切、PCR鉴定,测序证实。结果获得GST-pcDNA3重组克隆。结论:本实验获得Sj26GST重组克隆,为进一步研制核酸疫苗创造了条件。%Aim To amplify the gene of 26-Kilodalton glutathione S-transferases of Schistosoma japonicum and construct a recombinant eukaryotic expression vector. Methods One pair of primers was designed according to the sequence of GST. The gene fragment of Sj26GST was obtained by RT-PCR,then it was cloned into a vector pcDNA3. It was corroborated through restriction enzymes cleaving,PCR and sequencing. Results GST-pcDNA3 recombinant clone was constructed successfully. Conclusions Sj26GST is a promising candidate of vaccine for Schistosoma japonicum. GSTrecombinant clone was achieved in the experiment. It is prepared for further study for DNA vaccine.

  10. Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells.

    Science.gov (United States)

    Cox, Christopher R; Coburn, Phillip S; Gilmore, Michael S

    2005-02-01

    The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.

  11. Automation 2017

    CERN Document Server

    Zieliński, Cezary; Kaliczyńska, Małgorzata

    2017-01-01

    This book consists of papers presented at Automation 2017, an international conference held in Warsaw from March 15 to 17, 2017. It discusses research findings associated with the concepts behind INDUSTRY 4.0, with a focus on offering a better understanding of and promoting participation in the Fourth Industrial Revolution. Each chapter presents a detailed analysis of a specific technical problem, in most cases followed by a numerical analysis, simulation and description of the results of implementing the solution in a real-world context. The theoretical results, practical solutions and guidelines presented are valuable for both researchers working in the area of engineering sciences and practitioners looking for solutions to industrial problems. .

  12. Marketing automation

    Directory of Open Access Journals (Sweden)

    TODOR Raluca Dania

    2017-01-01

    Full Text Available The automation of the marketing process seems to be nowadays, the only solution to face the major changes brought by the fast evolution of technology and the continuous increase in supply and demand. In order to achieve the desired marketing results, businessis have to employ digital marketing and communication services. These services are efficient and measurable thanks to the marketing technology used to track, score and implement each campaign. Due to the technical progress, the marketing fragmentation, demand for customized products and services on one side and the need to achieve constructive dialogue with the customers, immediate and flexible response and the necessity to measure the investments and the results on the other side, the classical marketing approached had changed continue to improve substantially.

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

    Science.gov (United States)

    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

  14. Complex archaea that bridge the gap between prokaryotes and eukaryotes.

    Science.gov (United States)

    Spang, Anja; Saw, Jimmy H; Jørgensen, Steffen L; Zaremba-Niedzwiedzka, Katarzyna; Martijn, Joran; Lind, Anders E; van Eijk, Roel; Schleper, Christa; Guy, Lionel; Ettema, Thijs J G

    2015-05-14

    The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of 'Lokiarchaeota', a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic 'starter-kit' to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

  15. An epigenetic toolkit allows for diverse genome architectures in eukaryotes.

    Science.gov (United States)

    Maurer-Alcalá, Xyrus X; Katz, Laura A

    2015-12-01

    Genome architecture varies considerably among eukaryotes in terms of both size and structure (e.g. distribution of sequences within the genome, elimination of DNA during formation of somatic nuclei). The diversity in eukaryotic genome architectures and the dynamic processes are only possible due to the well-developed epigenetic toolkit, which probably existed in the Last Eukaryotic Common Ancestor (LECA). This toolkit may have arisen as a means of navigating the genomic conflict that arose from the expansion of transposable elements within the ancestral eukaryotic genome. This toolkit has been coopted to support the dynamic nature of genomes in lineages across the eukaryotic tree of life. Here we highlight how the changes in genome architecture in diverse eukaryotes are regulated by epigenetic processes, such as DNA elimination, genome rearrangements, and adaptive changes to genome architecture. The ability to epigenetically modify and regulate genomes has contributed greatly to the diversity of eukaryotes observed today.

  16. Semi-automated curation of protein subcellular localization: a text mining-based approach to Gene Ontology (GO Cellular Component curation

    Directory of Open Access Journals (Sweden)

    Chan Juancarlos

    2009-07-01

    Full Text Available Abstract Background Manual curation of experimental data from the biomedical literature is an expensive and time-consuming endeavor. Nevertheless, most biological knowledge bases still rely heavily on manual curation for data extraction and entry. Text mining software that can semi- or fully automate information retrieval from the literature would thus provide a significant boost to manual curation efforts. Results We employ the Textpresso category-based information retrieval and extraction system http://www.textpresso.org, developed by WormBase to explore how Textpresso might improve the efficiency with which we manually curate C. elegans proteins to the Gene Ontology's Cellular Component Ontology. Using a training set of sentences that describe results of localization experiments in the published literature, we generated three new curation task-specific categories (Cellular Components, Assay Terms, and Verbs containing words and phrases associated with reports of experimentally determined subcellular localization. We compared the results of manual curation to that of Textpresso queries that searched the full text of articles for sentences containing terms from each of the three new categories plus the name of a previously uncurated C. elegans protein, and found that Textpresso searches identified curatable papers with recall and precision rates of 79.1% and 61.8%, respectively (F-score of 69.5%, when compared to manual curation. Within those documents, Textpresso identified relevant sentences with recall and precision rates of 30.3% and 80.1% (F-score of 44.0%. From returned sentences, curators were able to make 66.2% of all possible experimentally supported GO Cellular Component annotations with 97.3% precision (F-score of 78.8%. Measuring the relative efficiencies of Textpresso-based versus manual curation we find that Textpresso has the potential to increase curation efficiency by at least 8-fold, and perhaps as much as 15-fold, given

  17. High genetic diversity and novelty in eukaryotic plankton assemblages inhabiting saline lakes in the Qaidam basin.

    Science.gov (United States)

    Wang, Jiali; Wang, Fang; Chu, Limin; Wang, Hao; Zhong, Zhiping; Liu, Zhipei; Gao, Jianyong; Duan, Hairong

    2014-01-01

    Saline lakes are intriguing ecosystems harboring extremely productive microbial communities in spite of their extreme environmental conditions. We performed a comprehensive analysis of the genetic diversity (18S rRNA gene) of the planktonic microbial eukaryotes (nano- and picoeukaryotes) in six different inland saline lakes located in the Qaidam Basin. The novelty level are high, with about 11.23% of the whole dataset showing 18S rRNA gene sequence reads obtained in investigated mesosaline lakes is closely related to Holozoa group (48.13%), whereas Stramenopiles (26.65%) and Alveolates (10.84%) are the next most common groups. Hypersaline lakes in the Qaidam Basin are also dominated by Holozoa group, accounting for 26.65% of the total number of sequence reads. Notably, Chlorophyta group are only found in high abundance in Lake Gasikule (28.00%), whereas less represented in other hypersaline lakes such as Gahai (0.50%) and Xiaochaidan (1.15%). Further analysis show that the compositions of planktonic eukaryotic assemblages are also most variable between different sampling sites in the same lake. Out of the parameters, four show significant correlation to this CCA: altitude, calcium, sodium and potassium concentrations. Overall, this study shows important gaps in the current knowledge about planktonic microbial eukaryotes inhabiting Qaidam Basin (hyper) saline water bodies. The identified diversity and novelty patterns among eukaryotic plankton assemblages in saline lake are of great importance for understanding and interpreting their ecology and evolution.

  18. Evolution of the vacuolar H sup + -ATPase: Implications for the origin of eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Gogarten, J.P.; Kibak, H.; Dittrich, P.; Taiz, L.; Bowman, E.J.; Bowman, B.J. (Univ. of California, Santa Cruz (USA)); Manolson, M.F.; Poole, R.J. (McGill Univ., Montreal, Quebec (Canada)); Date, Takayasu (Kanazawa Medical School, Ishikawa (Japan)); Oshima, Tairo; Konishi, Jin; Denda, Kimitoshi; Yoshida, Masasuke (Tokyo Institute of Technology, Yokohama (Japan))

    1989-09-01

    Active transport across the vaculoar components of the eukaryotic endomembrane system is energized by a specific vacuolar H{sup +}-ATPase. The amino acid sequences of the 70- and 60-kDa subunits of the vacuolar H{sup +}-ATPase are {approx}25% identical to the {beta} and {alpha} subunits, respectively, of the eubacterial-type F{sub 0}F{sub 1}-ATPases. The authors now report that the same vacuolar H{sup +}-ATPase subunits are {approx}50% identical to the {alpha} and {beta} subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the F{sub 0}F{sub 1}-ATPase {beta} subunit but is present in the {alpha} subunit of Sulfolobus. Since the two types of subunits are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H{sup +}-ATPase of eukaryotes arose by the internalization of the plasma membrane H{sup +}-ATPase of an archaebacterial-like ancestral cell.

  19. Construction of Eukaryotic Recombinant Expression Plasmid Containing gD Gene of Feline Herpesvirus Type 1 from Tiger%虎源传染性鼻气管炎病毒gD基因重组真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    张晓明; 胡俊; 彭仕明; 陈武

    2014-01-01

    The primers of gD genes of the FHV-1 were designed according to sequences of the FHV-1 from GenBank,gD gene was amplified by PCR and cloned successfully into pMD18-T Simple vector.gD gene was constructed into eukaryotic expression vector pcDNA 3.1(+),the recombinant plasmid ,named as pcDNA-gD,was transfected into 293T cells.The results of RT-PCR and Western blot indicated that the gD gene was transcripted and expressed,and the recombinant fusion protein was about 42.29 ku.The results lay an important foundation for the further research of FHV-1.%根据从虎气管组织扩增获得的猫传染性鼻气管炎病毒相关核酸序列,设计扩增编码虎源猫传染性鼻气管炎病毒 gD 蛋白基因片段的引物,通过 PCR 扩增出 gD 基因片段,并成功插入到 pMD18-T Sim-ple 载体中,筛选获得重组质粒,命名为18T-gD。重组质粒通过 Hin d Ⅲ和 Xho Ⅰ酶切后,插入 pcDNA 3.1(+)真核表达载体构建出 gD 基因的重组真核表达质粒 pcDNA-gD。用脂质体将重组真核表达质粒 pcD-NA-gD 转染293T 细胞,通过 RT-PCR 和 Western blot 检测,结果显示 gD 基因在真核细胞中得到正确转录和表达,所表达蛋白分子质量约为42.29 ku,为虎源猫传染性鼻气管炎基因疫苗的研究奠定了基础。

  20. Construction of human Gax gene eukaryotic expression vector and its expression in vascular smooth muscle cells%人Gax基因真核表达载体的构建及在血管平滑肌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    郑辉; 薛松; 连锋; 汪永义

    2011-01-01

    AIM: To construct human Gax eukaryotic expression vector of pEGFP-N1-Gax, and observe its expression in the rabbit vascular smooth muscle calls (VSMCs). METHODS: human Gax cDNA was obtained by polymerase chain reaction(PCR) from pCMV-SPORT6-Gax plasmid. After digested with Nhe l and Xho l, the PCR product was cloned into eukaryotic expression vector pEGFP-N1 of green fluorescent protein(GFP) reported gene encoding green fluorescence protein, and then the recombinant plasmid pEGFP-N1-Gax was transfected into rabbit VSMCs using Sofast after it was identified by restriction enzyme digestion analysis and gene sequencing. Human Gax expression in rabbit VSMCs was detected by examining GFP expression of transfected cells under fluorescence microscope and RT-PCR. RESULTS: Agarose gel electrophoresis detection showed that human Gax DNA segment was about 915 bp, which accorded with the expectation. The restriction enzyme digestion analysis and DNA sequencing assays of recombinant vector pEGFP-N1-Gax showed the correct orientation and sequence. The expression of GFP in rabbit VSMCs transfected with pEGFP-N1-Gax was observed by fluorescence microscopy, and the expression of human Gax mRNA was confirmed by RT-PCR. CONCLUSION; The recombinant plasmid pEGFP-N1-Gax is successfully constructed, and expressed positively in rabbit VSMCs, it provide experimental basis to study the effects of Gax gene on cardiovascular disease.%目的:构建人Gax基因真核表达载体,并观察在兔血管平滑肌细胞中的表达.方法:通过PCR从pCMV-SPORT6-Gax质粒中扩增出人Gax cDNA片段,经双酶切后装入到有绿色荧光蛋白报告基因的真核表达载体pEGFP-N1中,经限制性内切酶酶切分析和DNA测序鉴定后通过梭华-Sofast转染试剂介导重组质粒转染至兔血管平滑肌细胞中进行表达.通过荧光显微镜观察转染细胞的绿色荧光蛋白表达和RT-PCR扩增转染细胞的cDNA来鉴定Gax在兔血管平滑肌细胞中的表达.结果:琼

  1. Prokaryotes Versus Eukaryotes: Who is Hosting Whom?

    Science.gov (United States)

    Tellez, Guillermo

    2014-01-01

    Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals' actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a "forgotten organ," functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short-chain fatty acids), a process, which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system. Despite these important effects, the mechanisms by which the gut microbial community influences the host's biology remain almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes, which encourage us to postulate: who is hosting whom?

  2. Prokaryotes versus Eukaryotes: Who is hosting whom?

    Directory of Open Access Journals (Sweden)

    Guillermo eTellez

    2014-10-01

    Full Text Available Microorganisms represent the largest component of biodiversity in our world. For millions of years, prokaryotic microorganisms have functioned as a major selective force shaping eukaryotic evolution. Microbes that live inside and on animals outnumber the animals’ actual somatic and germ cells by an estimated 10-fold. Collectively, the intestinal microbiome represents a ‘forgotten organ’, functioning as an organ inside another that can execute many physiological responsibilities. The nature of primitive eukaryotes was drastically changed due to the association with symbiotic prokaryotes facilitating mutual coevolution of host and microbe. Phytophagous insects have long been used to test theories of evolutionary diversification; moreover, the diversification of a number of phytophagous insect lineages has been linked to mutualisms with microbes. From termites and honey bees to ruminants and mammals, depending on novel biochemistries provided by the prokaryotic microbiome, the association helps to metabolize several nutrients that the host cannot digest and converting these into useful end products (such as short chain fatty acids, a process which has huge impact on the biology and homeostasis of metazoans. More importantly, in a direct and/or indirect way, the intestinal microbiota influences the assembly of gut-associated lymphoid tissue, helps to educate immune system, affects the integrity of the intestinal mucosal barrier, modulates proliferation and differentiation of its epithelial lineages, regulates angiogenesis, and modifies the activity of enteric as well as the central nervous system,. Despite these important effects, the mechanisms by which the gut microbial community influences the host’s biology remains almost entirely unknown. Our aim here is to encourage empirical inquiry into the relationship between mutualism and evolutionary diversification between prokaryotes and eukaryotes which encourage us to postulate: Who is

  3. Searching for the role of protein phosphatases in eukaryotic microorganisms

    Directory of Open Access Journals (Sweden)

    da-Silva A.M.

    1999-01-01

    Full Text Available Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively. Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

  4. Asgard archaea illuminate the origin of eukaryotic cellular complexity.

    Science.gov (United States)

    Zaremba-Niedzwiedzka, Katarzyna; Caceres, Eva F; Saw, Jimmy H; Bäckström, Disa; Juzokaite, Lina; Vancaester, Emmelien; Seitz, Kiley W; Anantharaman, Karthik; Starnawski, Piotr; Kjeldsen, Kasper U; Stott, Matthew B; Nunoura, Takuro; Banfield, Jillian F; Schramm, Andreas; Baker, Brett J; Spang, Anja; Ettema, Thijs J G

    2017-01-19

    The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the 'Asgard' superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of 'eukaryote-specific' proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.

  5. Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex

    Science.gov (United States)

    2016-06-07

    DISCUSSION ABC is an important nosocomial pathogen seen with in- creasing frequency throughout medical facilities . The ability of ABC to acquire extensive...settings because of their efficiency and convenience . However, the accuracy of automated methods for testing the susceptibilities of ABC isolates to

  6. Dyneins across eukaryotes: a comparative genomic analysis.

    Science.gov (United States)

    Wickstead, Bill; Gull, Keith

    2007-12-01

    Dyneins are large minus-end-directed microtubule motors. Each dynein contains at least one dynein heavy chain (DHC) and a variable number of intermediate chains (IC), light intermediate chains (LIC) and light chains (LC). Here, we used genome sequence data from 24 diverse eukaryotes to assess the distribution of DHCs, ICs, LICs and LCs across Eukaryota. Phylogenetic inference identified nine DHC families (two cytoplasmic and seven axonemal) and six IC families (one cytoplasmic). We confirm that dyneins have been lost from higher plants and show that this is most likely because of a single loss of cytoplasmic dynein 1 from the ancestor of Rhodophyta and Viridiplantae, followed by lineage-specific losses of other families. Independent losses in Entamoeba mean that at least three extant eukaryotic lineages are entirely devoid of dyneins. Cytoplasmic dynein 2 is associated with intraflagellar transport (IFT), but in two chromalveolate organisms, we find an IFT footprint without the retrograde motor. The distribution of one family of outer-arm dyneins accounts for 2-headed or 3-headed outer-arm ultrastructures observed in different organisms. One diatom species builds motile axonemes without any inner-arm dyneins (IAD), and the unexpected conservation of IAD I1 in non-flagellate algae and LC8 (DYNLL1/2) in all lineages reveals a surprising fluidity to dynein function.

  7. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  8. Eukaryotic and Prokaryotic Cytoskeletons: Structure and Mechanics

    Science.gov (United States)

    Gopinathan, Ajay

    2013-03-01

    The eukaryotic cytoskeleton is an assembly of filamentous proteins and a host of associated proteins that collectively serve functional needs ranging from spatial organization and transport to the production and transmission of forces. These systems can exhibit a wide variety of non-equilibrium, self-assembled phases depending on context and function. While much recent progress has been made in understanding the self-organization, rheology and nonlinear mechanical properties of such active systems, in this talk, we will concentrate on some emerging aspects of cytoskeletal physics that are promising. One such aspect is the influence of cytoskeletal network topology and its dynamics on both active and passive intracellular transport. Another aspect we will highlight is the interplay between chirality of filaments, their elasticity and their interactions with the membrane that can lead to novel conformational states with functional implications. Finally we will consider homologs of cytoskeletal proteins in bacteria, which are involved in templating cell growth, segregating genetic material and force production, which we will discuss with particular reference to contractile forces during cell division. These prokaryotic structures function in remarkably similar yet fascinatingly different ways from their eukaryotic counterparts and can enrich our understanding of cytoskeletal functioning as a whole.

  9. Strong eukaryotic IRESs have weak secondary structure.

    Directory of Open Access Journals (Sweden)

    Xuhua Xia

    Full Text Available BACKGROUND: The objective of this work was to investigate the hypothesis that eukaryotic Internal Ribosome Entry Sites (IRES lack secondary structure and to examine the generality of the hypothesis. METHODOLOGY/PRINCIPAL FINDINGS: IRESs of the yeast and the fruit fly are located in the 5'UTR immediately upstream of the initiation codon. The minimum folding energy (MFE of 60 nt RNA segments immediately upstream of the initiation codons was calculated as a proxy of secondary structure stability. MFE of the reverse complements of these 60 nt segments was also calculated. The relationship between MFE and empirically determined IRES activity was investigated to test the hypothesis that strong IRES activity is associated with weak secondary structure. We show that IRES activity in the yeast and the fruit fly correlates strongly with the structural stability, with highest IRES activity found in RNA segments that exhibit the weakest secondary structure. CONCLUSIONS: We found that a subset of eukaryotic IRESs exhibits very low secondary structure in the 5'-UTR sequences immediately upstream of the initiation codon. The consistency in results between the yeast and the fruit fly suggests a possible shared mechanism of cap-independent translation initiation that relies on an unstructured RNA segment.

  10. Determination and inference of eukaryotic transcription factor sequence specificity.

    Science.gov (United States)

    Weirauch, Matthew T; Yang, Ally; Albu, Mihai; Cote, Atina G; Montenegro-Montero, Alejandro; Drewe, Philipp; Najafabadi, Hamed S; Lambert, Samuel A; Mann, Ishminder; Cook, Kate; Zheng, Hong; Goity, Alejandra; van Bakel, Harm; Lozano, Jean-Claude; Galli, Mary; Lewsey, Mathew G; Huang, Eryong; Mukherjee, Tuhin; Chen, Xiaoting; Reece-Hoyes, John S; Govindarajan, Sridhar; Shaulsky, Gad; Walhout, Albertha J M; Bouget, François-Yves; Ratsch, Gunnar; Larrondo, Luis F; Ecker, Joseph R; Hughes, Timothy R

    2014-09-11

    Transcription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif "library" can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

  11. Identification of selenocyst- eine insertion sequence (SECIS) element in eukaryotic selenoproteins by RNA Draw program

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The computer program RNA Draw was used to identify the secondary structures in the 3′ untranslated re- gions (3′UTRs) of the mRNAs from 46 eukaryotic seleno- proteins among 7 species. The program found one or two possible SECIS elements in these selenoproteins. The SECIS element consists of a stem-loop or hairpin structure with three conserved sequences of AUGA-(A)AA-GA. SECIS element was not found by the RNA Draw program in randomly selected non-selenoproteins. The results showed that SECIS element is the unique character of the genes of eukaryotic selenoproteins. Thus it is possible to use RNA Draw to search the SECIS elements in gene bank for poten- tial new selenoproteins.

  12. Construction of eukaryotic expression vector of Der p2 gene and its expression in mouse dendritic cells%Der p 2基因真核表达载体构建及其在小鼠树突状细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    毕玉田; 王彦; 吴奎; 王长征; 钱桂生

    2008-01-01

    背景:树突状细胞是目前已知的最强大的抗原提呈细胞,已经被应用于免疫治疗的研究中.目的:构建Derp 2真核表达载体,并证明能在小鼠骨髓来源的树突状细胞中表达.设计、时间及地点:单一样本观察,实验于2005-05/12在解放军第二军医大学新桥医院全军呼吸疾病研究所完成.材料:C57BL/6小鼠;plambd-Derp 2购自美国HESKA公司;pCI-neo质粒为解放军第三军医大学新桥医院全军呼吸疾病研究所保存.方法:体外分离,培养小鼠骨髓来源树突状细胞.将原核表达质粒plambdDer p 2携带的Derp2全长cDNA切下,重组到真核表达质粒pCI-neo中,然后在脂质体介导下,将重组质粒转染到小鼠骨髓来源的树突状细胞,以末转染质粒和转染空白载体pCl-neo的树突状细胞为对照.主要观察指标:①pCI-neoDer p 2重组质粒结构鉴定.②并用反转录-聚合酶链反应、Western Blot检测Der p 2mRNA和蛋白表达.结果:测序证实构建的重组质粒中携带了Der p2的全长cDNA序列,且与GeneBank序列完全一致.反转录-聚合酶链反应和Western Blot检测结果提示,重组质粒转染的树突状细胞能表达Der p 2 mRNA和Derp2蛋白.结论:成功构建重组Der p 2基因真核表达载体,其转染树突状细胞后,能有效地表达于树突状细胞中.%BACKGROUND: Dendritic cells, the most potent antigen presenting cells known at present, have been extensively used in the immunotherapy as adjuvant. OBJECTIVE: The present study was to construct Der p 2 eukaryotic expression vector and validate its expression in the mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA between May and December 2005. MATERIALS: C57BL/6 mice were included. Plambd-Der p 2 was the product of Heska Company, USA.pCI-neo plasmid was provided by the Institute of

  13. Cationic amphiphiles as delivery system for genes into eukaryotic cells

    NARCIS (Netherlands)

    Oberle, Volker; Zuhorn, Inge S.; Audouy, Sandrine; Bakowsky, Udo; Smisterová, Jarmila; Engberts, Jan B.F.N.; Hoekstra, Dick; Gregoriadis, G; McCormack, B

    2000-01-01

    Cationic liposomes, consisting of synthetic amphiphiles and a so-called helper lipid, rapidly form complexes with DNA, known as lipoplexes. When incubated with cells in culture, the DNA can be delivered into the cell and becomes expressed. Because of these properties, lipoplexes are considered a use

  14. High-resolution statistical mapping reveals gene territories in live yeast.

    Science.gov (United States)

    Berger, Axel B; Cabal, Ghislain G; Fabre, Emmanuelle; Duong, Tarn; Buc, Henri; Nehrbass, Ulf; Olivo-Marin, Jean-Christophe; Gadal, Olivier; Zimmer, Christophe

    2008-12-01

    The nonrandom positioning of genes inside eukaryotic cell nuclei is implicated in central nuclear functions. However, the spatial organization of the genome remains largely uncharted, owing to limited resolution of optical microscopy, paucity of nuclear landmarks and moderate cell sampling. We developed a computational imaging approach that creates high-resolution probabilistic maps of subnuclear domains occupied by individual loci in budding yeast through automated analysis of thousands of living cells. After validation, we applied the technique to genes involved in galactose metabolism and ribosome biogenesis. We found that genomic loci are confined to 'gene territories' much smaller than the nucleus, which can be remodeled during transcriptional activation, and that the nucleolus is an important landmark for gene positioning. The technique can be used to visualize and quantify territory positions relative to each other and to nuclear landmarks, and should advance studies of nuclear architecture and function.

  15. EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens.

    Science.gov (United States)

    Peng, Duo; Tarleton, Rick

    2015-10-01

    Recent development of CRISPR-Cas9 genome editing has enabled highly efficient and versatile manipulation of a variety of organisms and adaptation of the CRISPR-Cas9 system to eukaryotic pathogens has opened new avenues for studying these otherwise hard to manipulate organisms. Here we describe a webtool, Eukaryotic Pathogen gRNA Design Tool (EuPaGDT; available at http://grna.ctegd.uga.edu), which identifies guide RNA (gRNA) in input gene(s) to guide users in arriving at well-informed and appropriate gRNA design for many eukaryotic pathogens. Flexibility in gRNA design, accommodating unique eukaryotic pathogen (gene and genome) attributes and high-throughput gRNA design are the main features that distinguish EuPaGDT from other gRNA design tools. In addition to employing an array of known principles to score and rank gRNAs, EuPaGDT implements an effective on-target search algorithm to identify gRNA targeting multi-gene families, which are highly represented in these pathogens and play important roles in host-pathogen interactions. EuPaGDT also identifies and scores microhomology sequences flanking each gRNA targeted cut-site; these sites are often essential for the microhomology-mediated end joining process used for double-stranded break repair in these organisms. EuPaGDT also assists users in designing single-stranded oligonucleotides for homology directed repair. In batch processing mode, EuPaGDT is able to process genome-scale sequences, enabling preparation of gRNA libraries for large-scale screening projects.

  16. Circular permutation of a synthetic eukaryotic chromosome with the telomerator

    Science.gov (United States)

    Mitchell, Leslie A.; Boeke, Jef D.

    2014-01-01

    Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the “telomerator,” a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes. PMID:25378705

  17. Genetic diversity of eukaryotic microorganisms in Lake Taihu, a large shallow subtropical lake in china.

    Science.gov (United States)

    Chen, Meijun; Chen, Feizhou; Yu, Yang; Ji, Jian; Kong, Fanxiang

    2008-10-01

    We investigated the genetic diversity of eukaryotic microorganisms (0.8-20 microm) by sequencing cloned 18S rRNA genes in six genetic libraries constructed from six locations in Lake Taihu, a large shallow subtropical lake in China. Genetic libraries of eukaryotic ribosomal RNA were screened by restriction fragment length polymorphism (RFLP) analysis, and one clone representative of each RFLP pattern was partially sequenced. A total of 528 clones were clustered into 165 RFLP patterns and finally into 131 operational taxonomic unit (OTUs). Phylogenetic analysis revealed that each library included many unique OTUs, as well as members of distantly related phylogenetic groups. A majority of the clones were from alveolates, stramenopiles, cercozoa, cryptophytes, chlorophytes, and fungi, with members of choanoflagellida, euglenida, centroheliozoa, ancyromonadidae, ichthyosporea, and kathablepharid representing a minor fraction of the library. Six OTUs (15 clones) were not related to any known eukaryotic group. Canonical correspondence analysis suggested that the differences in eukaryotic microorganism community composition of in the six regions were partially related to trophic status, sediment resuspension, and top-down regulation by metazooplankton.

  18. EuMicroSatdb: A database for microsatellites in the sequenced genomes of eukaryotes

    Directory of Open Access Journals (Sweden)

    Grover Atul

    2007-07-01

    Full Text Available Abstract Background Microsatellites have immense utility as molecular markers in different fields like genome characterization and mapping, phylogeny and evolutionary biology. Existing microsatellite databases are of limited utility for experimental and computational biologists with regard to their content and information output. EuMicroSatdb (Eukaryotic MicroSatellite database http://ipu.ac.in/usbt/EuMicroSatdb.htm is a web based relational database for easy and efficient positional mining of microsatellites from sequenced eukaryotic genomes. Description A user friendly web interface has been developed for microsatellite data retrieval using Active Server Pages (ASP. The backend database codes for data extraction and assembly have been written using Perl based scripts and C++. Precise need based microsatellites data retrieval is possible using different input parameters like microsatellite type (simple perfect or compound perfect, repeat unit length (mono- to hexa-nucleotide, repeat number, microsatellite length and chromosomal location in the genome. Furthermore, information about clustering of different microsatellites in the genome can also be retrieved. Finally, to facilitate primer designing for PCR amplification of any desired microsatellite locus, 200 bp upstream and downstream sequences are provided. Conclusion The database allows easy systematic retrieval of comprehensive information about simple and compound microsatellites, microsatellite clusters and their locus coordinates in 31 sequenced eukaryotic genomes. The information content of the database is useful in different areas of research like gene tagging, genome mapping, population genetics, germplasm characterization and in understanding microsatellite dynamics in eukaryotic genomes.

  19. Arsenic transport in prokaryotes and eukaryotic microbes.

    Science.gov (United States)

    Rosen, Barry P; Tamás, Markus J

    2010-01-01

    Aquaporins (AQPs) and aquaglyceroporins facilitate transport of a broad spectrum of substrates such as water, glycerol and other small uncharged solutes. More recently, AQPs ave also been shown to facilitate diffusion of metalloids such as arsenic (As) and antimony (Sb). At neutral pH, the trivalent forms of these metalloids are structurally similar to glycerol and hence they can enter cells through AQPs. As- and Sb-containing compounds are toxic to cells, yet both metalloids are used as chemotherapeutic agents for treating acute promyelocytic leukemia and diseases caused by protozoan parasites. In this chapter, we will review the role of AQPs and other proteins in metalloid transport in prokaryotes and eukaryotic microbes.

  20. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  1. Regulation of Eukaryotic Initiation Factor 4E and Its Isoform: Implications for Antiviral Strategy in Plants

    Institute of Scientific and Technical Information of China (English)

    Yu-Yang Zhang; Han-Xia Li; Bo Ou-yang; Zhi-Biao Ye

    2006-01-01

    In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomicaiiy important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotic initiation factor 4E (elF4E). elF4E is one of the most important translation initiation factors involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by elF4E and its isoform elF (iso)4E occurs in several plant-virus interactions, thus indicating a potential new role for elF4E/elL(iso)4E in resistance strategies against plant viruses. In this review, we briefly describe elF4E activity in plant translation, its potential role, and functions of the elF4E subfamily in plant-virus interactions. Other initiation factors such as elF4G could also play a role in plant resistance against viruses. Finally, the potential for developing elF4E-medlated resistance to plant viruses in the future is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by elF4E. Knowledge of a particular plant-virus interaction will help to deepen our understanding of elF4E and other eukaryotic initiation factors, and their involvement in virus disease control.

  2. Eukaryotic protein production in designed storage organelles

    Directory of Open Access Journals (Sweden)

    Saloheimo Markku

    2009-01-01

    Full Text Available Abstract Background Protein bodies (PBs are natural endoplasmic reticulum (ER or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells. Results Various Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain. Conclusion The Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.

  3. How and why DNA barcodes underestimate the diversity of microbial eukaryotes.

    Directory of Open Access Journals (Sweden)

    Gwenael Piganeau

    Full Text Available BACKGROUND: Because many picoplanktonic eukaryotic species cannot currently be maintained in culture, direct sequencing of PCR-amplified 18S ribosomal gene DNA fragments from filtered sea-water has been successfully used to investigate the astounding diversity of these organisms. The recognition of many novel planktonic organisms is thus based solely on their 18S rDNA sequence. However, a species delimited by its 18S rDNA sequence might contain many cryptic species, which are highly differentiated in their protein coding sequences. PRINCIPAL FINDINGS: Here, we investigate the issue of species identification from one gene to the whole genome sequence. Using 52 whole genome DNA sequences, we estimated the global genetic divergence in protein coding genes between organisms from different lineages and compared this to their ribosomal gene sequence divergences. We show that this relationship between proteome divergence and 18S divergence is lineage dependent. Unicellular lineages have especially low 18S divergences relative to their protein sequence divergences, suggesting that 18S ribosomal genes are too conservative to assess planktonic eukaryotic diversity. We provide an explanation for this lineage dependency, which suggests that most species with large effective population sizes will show far less divergence in 18S than protein coding sequences. CONCLUSIONS: There is therefore a trade-off between using genes that are easy to amplify in all species, but which by their nature are highly conserved and underestimate the true number of species, and using genes that give a better description of the number of species, but which are more difficult to amplify. We have shown that this trade-off differs between unicellular and multicellular organisms as a likely consequence of differences in effective population sizes. We anticipate that biodiversity of microbial eukaryotic species is underestimated and that numerous "cryptic species" will become

  4. Manufacturing and automation

    Directory of Open Access Journals (Sweden)

    Ernesto Córdoba Nieto

    2010-04-01

    Full Text Available The article presents concepts and definitions from different sources concerning automation. The work approaches automation by virtue of the author’s experience in manufacturing production; why and how automation prolects are embarked upon is considered. Technological reflection regarding the progressive advances or stages of automation in the production area is stressed. Coriat and Freyssenet’s thoughts about and approaches to the problem of automation and its current state are taken and examined, especially that referring to the problem’s relationship with reconciling the level of automation with the flexibility and productivity demanded by competitive, worldwide manufacturing.

  5. 不可分型流感嗜血杆菌外膜蛋白P6基因真核质粒的保护性研究%Research on the protective immunity of the eukaryotic vector for the gene encoding the outer membrane protein P6 of the nontypeable Haemophilus influenzae

    Institute of Scientific and Technical Information of China (English)

    何多姣; 张彦霞; 刘雪晴; 李双霞; 贾天军; 张玉妥

    2011-01-01

    目的:探讨不可分型流感嗜血杆茵(nontypeable Haemophilus influenzae,NTHi)外膜蛋白P6(outer membrane protein P6,P6)真核质粒的免疫保护性及其可能的保护机制.方法:构建pcDNA3.1/HisA-P6核酸疫苗,转染HeLa细胞;将P6质粒免疫BALB/c小鼠后,ELISA法检测小鼠脾淋巴细胞IFN-γ和IL-4产生水平:CCK-8法分析脾淋巴细胞增殖情况,并建立小鼠NTHi感染模型,检测小鼠鼻咽部灌洗液NTHi数目变化:HE染色法分析鼻黏膜病理变化.结果:pcDNA3.1/HisA-P6在HeLa细胞中有目的蛋白的表达,免疫后pcDNA3.1/HisA-P6组小鼠脾淋巴细胞产生的IFN-γ及刺激指数均高于pcDNA3.1/HisA、PBS对照组(P<0.05).而IL-4的表达无差异;P6组鼻咽部NTHi清除率也显著高于对照组(P<0.01);病理显示P6组小鼠鼻黏膜组织结构基本正常,而时照组鼻黏膜紊乱、脱落.讨论:P6核酸疫苗可诱导小鼠产生明显的抗NTHi保护效应,其可能的保护机制包括细胞免疫、体液免疫及黏膜免疫.%Objective To discuss the protective immunity of the eukaryotic vector for the gene encoding the outer memhrane protein P6 (P6)of the nontypeahle Haemophilus influenzae (NTHi) and its possible protective mechanism. Methods pcDNA3.1/HisA-P6 was constructed and transfected into HeLa cells.Then the plasmids was used to immunize BALB/c mice,IFN-γ and IL-4 producted by spleen lymphocyte was detected by ELISA ; The proliferation of spleen lymphocyte was analysed by CCK-8 ; The NTHi infection model was also established . then the nasopharyngeal clearance of NTHi and the nasal mucosa pathological changes were assessed. Results The mice spleen lymphocyte immunized with pcDNA3.1/HisA-P6 evoked a higher production of IFN-γ and stimulation index than controls which were immunized by pcDNA3.1/HisA or PBS (P < 0.05) . while there were no significant difference on IL-4 among them ; The nasopharyngeal clearance of NTHi immunized with pcDNA3.1/HisA-P6 was also higher than controls ( P

  6. Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis.

    Science.gov (United States)

    Wang, Yong; Tian, Ren Mao; Gao, Zhao Ming; Bougouffa, Salim; Qian, Pei-Yuan

    2014-01-01

    Eukaryotic 18S ribosomal RNA (rRNA) gene primers that feature a wide coverage are critical in detecting the composition of eukaryotic microscopic organisms in ecosystems. Here, we predicted 18S rRNA primers based on consecutive conserved sites and evaluated their coverage efficiency and scope of application to different eukaryotic groups. After evaluation, eight of them were considered as qualified 18S primers based on coverage rate. Next, we examined common conserved regions in prokaryotic 16S and eukaryotic 18S rRNA sequences to design 16S/18S universal primers. Three 16S/18S candidate primers, U515, U1390 and U1492, were then considered to be suitable for simultaneous amplification of the rRNA sequences in three domains. Eukaryotic 18S and prokaryotic 16S rRNA genes in a sponge were amplified simultaneously using universal primers U515 and U1390, and the subsequent sorting of pyrosequenced reads revealed some distinctive communities in different parts of the sample. The real difference in biodiversity between prokaryotic and eukaryotic symbionts could be discerned as the dissimilarity between OTUs was increased from 0.005 to 0.1. A network of the communities in external and internal parts of the sponge illustrated the co-variation of some unique microbes in certain parts of the sponge, suggesting that the universal primers are useful in simultaneous detection of prokaryotic and eukaryotic microbial communities.

  7. Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Eukaryotic 18S ribosomal RNA (rRNA gene primers that feature a wide coverage are critical in detecting the composition of eukaryotic microscopic organisms in ecosystems. Here, we predicted 18S rRNA primers based on consecutive conserved sites and evaluated their coverage efficiency and scope of application to different eukaryotic groups. After evaluation, eight of them were considered as qualified 18S primers based on coverage rate. Next, we examined common conserved regions in prokaryotic 16S and eukaryotic 18S rRNA sequences to design 16S/18S universal primers. Three 16S/18S candidate primers, U515, U1390 and U1492, were then considered to be suitable for simultaneous amplification of the rRNA sequences in three domains. Eukaryotic 18S and prokaryotic 16S rRNA genes in a sponge were amplified simultaneously using universal primers U515 and U1390, and the subsequent sorting of pyrosequenced reads revealed some distinctive communities in different parts of the sample. The real difference in biodiversity between prokaryotic and eukaryotic symbionts could be discerned as the dissimilarity between OTUs was increased from 0.005 to 0.1. A network of the communities in external and internal parts of the sponge illustrated the co-variation of some unique microbes in certain parts of the sponge, suggesting that the universal primers are useful in simultaneous detection of prokaryotic and eukaryotic microbial communities.

  8. Causes and consequences of eukaryotization through mutualistic endosymbiosis and compartmentalization

    NARCIS (Netherlands)

    Hengeveld, R.; Fedonkin, M.A.

    2004-01-01

    This paper reviews and extends ideas of eukaryotization by endosymbiosis. These ideas are put within an historical context of processes that may have led up to eukaryotization and those that seem to have resulted from this process. Our starting point for considering the emergence and development of

  9. Molecular paleontology and complexity in the last eukaryotic common ancestor.

    Science.gov (United States)

    Koumandou, V Lila; Wickstead, Bill; Ginger, Michael L; van der Giezen, Mark; Dacks, Joel B; Field, Mark C

    2013-01-01

    Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.

  10. Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: implications for health risks

    Science.gov (United States)

    The goal of this study was to characterize microbial eukaryotes over a 12 month period, so as to provide insight into the occurrence of potentially important predators and bacterial hosts in hot and cold premise plumbing. Nearly 6,300 partial (600 bp) 18S rRNA gene sequences from...

  11. An automated swimming respirometer

    DEFF Research Database (Denmark)

    STEFFENSEN, JF; JOHANSEN, K; BUSHNELL, PG

    1984-01-01

    An automated respirometer is described that can be used for computerized respirometry of trout and sharks.......An automated respirometer is described that can be used for computerized respirometry of trout and sharks....

  12. Configuration Management Automation (CMA) -

    Data.gov (United States)

    Department of Transportation — Configuration Management Automation (CMA) will provide an automated, integrated enterprise solution to support CM of FAA NAS and Non-NAS assets and investments. CMA...

  13. Autonomy and Automation

    Science.gov (United States)

    Shively, Jay

    2017-01-01

    A significant level of debate and confusion has surrounded the meaning of the terms autonomy and automation. Automation is a multi-dimensional concept, and we propose that Remotely Piloted Aircraft Systems (RPAS) automation should be described with reference to the specific system and task that has been automated, the context in which the automation functions, and other relevant dimensions. In this paper, we present definitions of automation, pilot in the loop, pilot on the loop and pilot out of the loop. We further propose that in future, the International Civil Aviation Organization (ICAO) RPAS Panel avoids the use of the terms autonomy and autonomous when referring to automated systems on board RPA. Work Group 7 proposes to develop, in consultation with other workgroups, a taxonomy of Levels of Automation for RPAS.

  14. MetWAMer: eukaryotic translation initiation site prediction

    Directory of Open Access Journals (Sweden)

    Brendel Volker

    2008-09-01

    Full Text Available Abstract Background Translation initiation site (TIS identification is an important aspect of the gene annotation process, requisite for the accurate delineation of protein sequences from transcript data. We have developed the MetWAMer package for TIS prediction in eukaryotic open reading frames of non-viral origin. MetWAMer can be used as a stand-alone, third-party tool for post-processing gene structure annotations generated by external computational programs and/or pipelines, or directly integrated into gene structure prediction software implementations. Results MetWAMer currently implements five distinct methods for TIS prediction, the most accurate of which is a routine that combines weighted, signal-based translation initiation site scores and the contrast in coding potential of sequences flanking TISs using a perceptron. Also, our program implements clustering capabilities through use of the k-medoids algorithm, thereby enabling cluster-specific TIS parameter utilization. In practice, our static weight array matrix-based indexing method for parameter set lookup can be used with good results in data sets exhibiting moderate levels of 5'-complete coverage. Conclusion We demonstrate that improvements in statistically-based models for TIS prediction can be achieved by taking the class of each potential start-methionine into account pending certain testing conditions, and that our perceptron-based model is suitable for the TIS identification task. MetWAMer represents a well-documented, extensible, and freely available software system that can be readily re-trained for differing target applications and/or extended with existing and novel TIS prediction methods, to support further research efforts in this area.

  15. Diversity and distribution of microbial eukaryotes in the deep tropical and subtropical North Atlantic Ocean

    Science.gov (United States)

    Morgan-Smith, Danielle; Clouse, Melissa A.; Herndl, Gerhard J.; Bochdansky, Alexander B.

    2013-08-01

    Employing a combination of 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate (DAPI-FITC) staining and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we distinguished a variety of taxonomic and morphological types of eukaryotic microbes in the central and deep water masses of the tropical and subtropical North Atlantic Ocean. Samples were taken along a transect across the tropical Atlantic, along the equatorial upwelling and into the West-African upwelling region. Samples were collected as deep as 7000 m in the Romanche Fracture Zone within the Mid-Atlantic Ridge. Approximately 50-70% of FISH-identified eukaryotes in deep water masses belong to one of seven groups: kinetoplastids, labyrinthulomycetes, fungi, diplonemids, group II alveolates, MAST 4 (stramenopiles), and an unidentified organism with a peculiar nuclear morphology. A smaller percentage of total eukaryotes was identified in the Central Water, especially in the oxygen minimum zone, than in deep water masses. CARD-FISH probes designed to identify broad taxonomic groups revealed kinetoplastids and fungi were more abundant than noted in previous studies employing 18S rRNA gene clone libraries. Group II alveolates, in contrast, were much less prevalent than previously reported. On a second survey, eukaryotic microbes were enumerated in the deep-sea basins below the North Atlantic subtropical gyre including the Vema Fracture Zone, which is another prominent trench in the Mid-Atlantic Ridge. The abundance of eukaryotes and chlorophyll concentrations were significantly different between the two cruises, which covered very different hydrographic regimes with associated high and low levels of primary production, respectively.

  16. Genetic diversity of eukaryotic plankton assemblages in Eastern Tibetan Lakes differing by their salinity and altitude.

    Science.gov (United States)

    Wu, Qinglong L; Chatzinotas, Antonis; Wang, Jianjun; Boenigk, Jens

    2009-10-01

    Eukaryotic plankton assemblages in 11 high-mountain lakes located at altitudes of 2,817 to 5,134 m and over a total area of ca. one million square kilometers on the Eastern Tibet Plateau, spanning a salinity gradient from 0.2 (freshwater) to 187.1 g l(-1) (hypersaline), were investigated by cultivation independent methods. Two 18S rRNA gene-based fingerprint approaches, i.e., the terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing were applied. Samples of the same lake type (e.g., freshwater) generally shared more of the same bands or T-RFs than samples of different types (e.g., freshwater versus saline). However, a certain number of bands or T-RFs among the samples within each lake were distinct, indicating the potential presence of significant genetic diversity within each lake. PCA indicated that the most significant environmental gradient among the investigated lakes was salinity. The observed molecular profiles could be further explained (17-24%) by ion percentage of chloride, carbonate and bicarbonate, and sulfate, which were also covaried with change of altitude and latitude. Sequence analysis of selected major DGGE bands revealed many sequences (largely protist) that are not related to any known cultures but to uncultured eukaryotic picoplankton and unidentified eukaryotes. One fourth of the retrieved sequences showed eukaryotic plankton, which were found worldwide and detected in low land lakes, were also detected in habitats located above 4,400 m, suggesting a cosmopolitan distribution of these phylotypes. Collectively, our study suggests that there was a high beta-diversity of eukaryotic plankton assemblages in the investigated Tibetan lakes shaped by multiple geographic and environmental factors.

  17. Microbial eukaryote plankton communities of high-mountain lakes from three continents exhibit strong biogeographic patterns.

    Science.gov (United States)

    Filker, Sabine; Sommaruga, Ruben; Vila, Irma; Stoeck, Thorsten

    2016-05-01

    Microbial eukaryotes hold a key role in aquatic ecosystem functioning. Yet, their diversity in freshwater lakes, particularly in high-mountain lakes, is relatively unknown compared with the marine environment. Low nutrient availability, low water temperature and high ultraviolet radiation make most high-mountain lakes extremely challenging habitats for life and require specific molecular and physiological adaptations. We therefore expected that these ecosystems support a plankton diversity that differs notably from other freshwater lakes. In addition, we hypothesized that the communities under study exhibit geographic structuring. Our rationale was that geographic dispersal of small-sized eukaryotes in high-mountain lakes over continental distances seems difficult. We analysed hypervariable V4 fragments of the SSU rRNA gene to compare the genetic microbial eukaryote diversity in high-mountain lakes located in the European Alps, the Chilean Altiplano and the Ethiopian Bale Mountains. Microbial eukaryotes were not globally distributed corroborating patterns found for bacteria, multicellular animals and plants. Instead, the plankton community composition emerged as a highly specific fingerprint of a geographic region even on higher taxonomic levels. The intraregional heterogeneity of the investigated lakes was mirrored in shifts in microbial eukaryote community structure, which, however, was much less pronounced compared with interregional beta-diversity. Statistical analyses revealed that on a regional scale, environmental factors are strong predictors for plankton community structures in high-mountain lakes. While on long-distance scales (>10 000 km), isolation by distance is the most plausible scenario, on intermediate scales (up to 6000 km), both contemporary environmental factors and historical contingencies interact to shift plankton community structures.

  18. Phylogeny of choanozoa, apusozoa, and other protozoa and early eukaryote megaevolution.

    Science.gov (United States)

    Cavalier-Smith, Thomas; Chao, Ema E-Y

    2003-05-01

    The primary diversification of eukaryotes involved protozoa, especially zooflagellates-flagellate protozoa without plastids. Understanding the origins of the higher eukaryotic kingdoms (two purely heterotrophic, Animalia and Fungi, and two primarily photosynthetic, Plantae and Chromista) depends on clarifying evolutionary relationships among the phyla of the ancestral kingdom Protozoa. We therefore sequenced 18S rRNA genes from 10 strains from the protozoan phyla Choanozoa and Apusozoa. Eukaryote diversity is encompassed by three early-radiating, arguably monophyletic groups: Amoebozoa, opisthokonts, and bikonts. Our taxon-rich rRNA phylogeny for eukaryotes allowing for intersite rate variation strongly supports the opisthokont clade (animals, Choanozoa, Fungi). It agrees with the view that Choanozoa are sisters of or ancestral to animals and reveals a novel nonflagellate choanozoan lineage, Ministeriida, sister either to choanoflagellates, traditionally considered animal ancestors, or to animals. Maximum likelihood trees suggest that within animals Placozoa are derived from medusozoan Cnidaria (we therefore place Placozoa as a class within subphylum Medusozoa of the Cnidaria) and hexactinellid sponges evolved from demosponges. The bikont and amoebozoan radiations are both very ill resolved. Bikonts comprise the kingdoms Plantae and Chromista and three major protozoan groups: alveolates, excavates, and Rhizaria. Our analysis weakly suggests that Apusozoa, represented by Ancyromonas and the apusomonads ( Apusomonas and the highly diverse and much more ancient genus Amastigomonas, from which it evolved), are not closely related to other Rhizaria and may be the most divergent bikont lineages. Although Ancyromonas and apusomonads appear deeply divergent in 18S rRNA trees, the trees neither refute nor support the monophyly of Apusozoa. The bikont phylum Cercozoa weakly but consistently appears as sister to Retaria (Foraminifera; Radiolaria), together forming a hitherto

  19. Workflow automation architecture standard

    Energy Technology Data Exchange (ETDEWEB)

    Moshofsky, R.P.; Rohen, W.T. [Boeing Computer Services Co., Richland, WA (United States)

    1994-11-14

    This document presents an architectural standard for application of workflow automation technology. The standard includes a functional architecture, process for developing an automated workflow system for a work group, functional and collateral specifications for workflow automation, and results of a proof of concept prototype.

  20. Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium.

    Science.gov (United States)

    Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2003-01-01

    Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.

  1. 猪TPST1基因的克隆、序列分析及真核表达载体的构建%Cloning, sequence characterization and construction of eukaryotic cell expression vector of TPST1 gene in pig

    Institute of Scientific and Technical Information of China (English)

    别墅; 孙洪涛; 张冬杰; 汪亮; 刘娣

    2011-01-01

    Ctyrosine O-sulfation is a common posttranslational modification of proteins which is mediated tyrosylprotein sulfotransferase. In this study, Porcine TPST1 cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR), using in silico cloning strategy based on porcine ESTs database, the nucleotide and amino acid sequences were characterized with bioinformatics methods. The length of the cloned porcine TPST1 cDNA was 1 543 bp including a 1 113 bp entire open reading frame (ORF), which encoded a 370 amino acids protein. Multiple sequences alignment and phylogeny analysis indicated that porcine TPST1 protein shared high similarity with that of other species, such as Bos taurus (97.6%), Musmusculus (96.8%), Rattus norvegicus (97%) and Homo sapiens. Protein sequence analysis showed that the deduced porcine TPST1 protein sequence (pl=9.0, MW= 42.13 ku). This study had completed the clone TPST1 of min pig and construction of eukaryotic cell expression vector of pcDNA3.1(+)-TPST1, and providing the theoretical basis for the expression and function research of TPST1 gene in cell.%酪氨酸硫化转移酶(Tyrosylrotein sulfotransferase,TPST)在酪氨酸硫化修饰过程中起调节作用.试验参照人、鼠、牛等动物的TPST1 mRNA全长序列设计引物,通过电子克隆结合RT-RCR的方法首次克隆获得包括完整CDS区的猪TPST1基因cDNA序列(HQ439987),分析其核苷酸序列及其编码蛋白质分子结构特征.结果表明,猪TPST1序列全长1543 bp,包含1113bp的完整开放阅读框,编码370个氨基酸.多序列比对和系统进化分析表明,该基因编码蛋白质与牛(97.6%)、小鼠(96.8%)、大鼠(97%)、人(97.03%)等物种TPST1蛋白质具有较高的一致性.蛋白质序列预测分析发现,猪TPST1氨基酸序列理论等电点(pI)为9.0,相对分子质量(MW)为42.13 ku.

  2. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  3. Visualizing Patterns of Marine Eukaryotic Diversity from Metabarcoding Data Using QIIME.

    Science.gov (United States)

    Leray, Matthieu; Knowlton, Nancy

    2016-01-01

    PCR amplification followed by deep sequencing of homologous gene regions is increasingly used to characterize the diversity and taxonomic composition of marine eukaryotic communities. This approach may generate millions of sequences for hundreds of samples simultaneously. Therefore, tools that researchers can use to visualize complex patterns of diversity for these massive datasets are essential. Efforts by microbiologists to understand the Earth and human microbiomes using high-throughput sequencing of the 16S rRNA gene has led to the development of several user-friendly, open-source software packages that can be similarly used to analyze eukaryotic datasets. Quantitative Insights Into Microbial Ecology (QIIME) offers some of the most helpful data visualization tools. Here, we describe functionalities to import OTU tables generated with any molecular marker (e.g., 18S, COI, ITS) and associated metadata into QIIME. We then present a range of analytical tools implemented within QIIME that can be used to obtain insights about patterns of alpha and beta diversity for marine eukaryotes.

  4. Eukaryotic checkpoints are absent in the cell division cycle of Entamoeba histolytica

    Indian Academy of Sciences (India)

    Sulagna Banerjee; Suchismita Das; Anuradha Lohia

    2002-11-01

    Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite, Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered in E. histolytica.

  5. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  6. GenColors-based comparative genome databases for small eukaryotic genomes.

    Science.gov (United States)

    Felder, Marius; Romualdi, Alessandro; Petzold, Andreas; Platzer, Matthias; Sühnel, Jürgen; Glöckner, Gernot

    2013-01-01

    Many sequence data repositories can give a quick and easily accessible overview on genomes and their annotations. Less widespread is the possibility to compare related genomes with each other in a common database environment. We have previously described the GenColors database system (http://gencolors.fli-leibniz.de) and its applications to a number of bacterial genomes such as Borrelia, Legionella, Leptospira and Treponema. This system has an emphasis on genome comparison. It combines data from related genomes and provides the user with an extensive set of visualization and analysis tools. Eukaryote genomes are normally larger than prokaryote genomes and thus pose additional challenges for such a system. We have, therefore, adapted GenColors to also handle larger datasets of small eukaryotic genomes and to display eukaryotic gene structures. Further recent developments include whole genome views, genome list options and, for bacterial genome browsers, the display of horizontal gene transfer predictions. Two new GenColors-based databases for two fungal species (http://fgb.fli-leibniz.de) and for four social amoebas (http://sacgb.fli-leibniz.de) were set up. Both new resources open up a single entry point for related genomes for the amoebozoa and fungal research communities and other interested users. Comparative genomics approaches are greatly facilitated by these resources.

  7. Waardenburg综合征致病基因SOX10重组真核细胞表达质粒的构建、表达及意义%Construction and analysis of recombinant eukaryotic expression plasmids for SOX10, the causative gene of Warrdenburg syndrome

    Institute of Scientific and Technical Information of China (English)

    张华; 冯娟; 陈红胜; 李家大; 罗浑金; 冯永

    2015-01-01

    Objective To study the exogenous expression and subcellular localization of wild type (WT) and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome (WS).Methods The plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag,which was as a template to generate expression plasmids for novel mutations G37fs,G38fs and E248fs of the SOX10 gene.The constructs were verified by direct sequencing.NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10,G37fs,G38fs and E248fs,respectively.The exogenous expression of WT SOX10 protein and mutant G37fs,G38fs and E248fs proteins were analyzed using Western blot assay,while their subcellular distribution were observed with an immunofluorescence assay.Results The DNA sequences of expression plasmids for SOX10 and its mutant G37fs,G38fs and E248f were all correct.Both WT and mutant SOX10 proteins were detected at the expected site.WT SOX10 and E248fs proteins have only localized in the nucleus,whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus.Conclusion Recombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed.Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins,which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.%目的 通过构建基因SOX10及其突变体表达质粒初步研究其外源性表达和定位表达,为研究Waardenburg综合征(Waardenburg syndrome,WS)发病机制提供实验基础.方法 通过分子克隆技术双酶切pECE-SOX10和pCMV-Flag后连接构建SOX10基因重组真核细胞表达质粒pCMV-SOX10-Flag,以其为模板分别构建SOX10

  8. Eukaryotic versus prokaryotic marine picoplankton ecology.

    Science.gov (United States)

    Massana, Ramon; Logares, Ramiro

    2013-05-01

    Marine microorganisms contribute markedly to global biomass and ecosystem function. They include a diverse collection of organisms differing in cell size and in evolutionary history. In particular, microbes within the picoplankton are similar in size but belong to two drastically different cellular plans, the prokaryotes and the eukaryotes. Compared with larger organisms, prokaryotes and picoeukaryotes share ecological features, such as high specific activity, large and constant abundances, and high dispersal potential. Still, there are some aspects where their different cell organization influences their ecological performance. First, prokaryotes have a huge metabolic versatility and are involved in all biogeochemical cycles, whereas picoeukaryotes are metabolically less flexible but can exploit diverse predatory life strategies due to their phagocytic capacity. Second, sexual reproduction is absent in prokaryotes but may be present in picoeukaryotes, thus determining different evolutionary diversification dynamics and making species limits clearer in picoeukaryotes. Finally, it is plausible that picoeukaryotes are less flexible to enter a reversible state of low metabolic activity, thus picoeukaryote assemblages may have fewer rare species and may be less resilient to environmental change. In summary, lumping together pico-sized microbes may be convenient for some ecological studies, but it is also important to keep in mind their differences.

  9. A new inhibitor of apoptosis from vaccinia virus and eukaryotes.

    Directory of Open Access Journals (Sweden)

    Caroline Gubser

    2007-02-01

    Full Text Available A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein. Stable expression of both viral GAAP (v-GAAP and human GAAP (h-GAAP, which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses.

  10. THE COMPLEX ORGANIZATION OF EUKARYOTIC CELL NUCLEUS: THE NUCLEAR BODIES (I

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    Cristian Campeanu

    2012-10-01

    Full Text Available Identified short time after the discovery of cells, over 300 years ago, the cell nucleus of eukaryotes continuously focused the interest of scientists, which used increasingly sophisticated research tools to clarify its complex structure and functions. The results of all these studies, especially those carried out in the second half of the past century, proved and confirmed that the eukaryotic cell nucleus is the control center of all cellular activities and also ensures the continuity of genetic information along successive generations of cells. These vital functions are the result of selective expression of genes contained in the nuclear chromatin, which is a high ordered and dynamic structure, in permanent and bilateral relations with other nuclear components. Based on these considerations, the present review aims to synthetize the latest researches and concepts about the cell nuclear territory in three distinctive parts, according to the complexity of the topic

  11. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

    Science.gov (United States)

    Guo, Junjie U.; Bartel, David P.

    2017-01-01

    In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in post-transcriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex–forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex–forming sequences. PMID:27708011

  12. The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups.

    Science.gov (United States)

    Koonin, Eugene V; Wolf, Yuri I; Nagasaki, Keizo; Dolja, Valerian V

    2008-12-01

    The recent discovery of RNA viruses in diverse unicellular eukaryotes and developments in evolutionary genomics have provided the means for addressing the origin of eukaryotic RNA viruses. The phylogenetic analyses of RNA polymerases and helicases presented in this Analysis article reveal close evolutionary relationships between RNA viruses infecting hosts from the Chromalveolate and Excavate supergroups and distinct families of picorna-like viruses of plants and animals. Thus, diversification of picorna-like viruses probably occurred in a 'Big Bang' concomitant with key events of eukaryogenesis. The origins of the conserved genes of picorna-like viruses are traced to likely ancestors including bacterial group II retroelements, the family of HtrA proteases and DNA bacteriophages.

  13. Automation in Clinical Microbiology

    Science.gov (United States)

    Ledeboer, Nathan A.

    2013-01-01

    Historically, the trend toward automation in clinical pathology laboratories has largely bypassed the clinical microbiology laboratory. In this article, we review the historical impediments to automation in the microbiology laboratory and offer insight into the reasons why we believe that we are on the cusp of a dramatic change that will sweep a wave of automation into clinical microbiology laboratories. We review the currently available specimen-processing instruments as well as the total laboratory automation solutions. Lastly, we outline the types of studies that will need to be performed to fully assess the benefits of automation in microbiology laboratories. PMID:23515547

  14. [Loop organization of eukaryotic chromosomes and triple-stranded DNA structures].

    Science.gov (United States)

    Glazkov, M V

    2011-01-01

    To study possible involvement of polypurine and polypyrimidine DNA tracks potentially capable to form triple-stranded structures (H-form of DNA) in compactization of eukaryotic chromosomes a search in silico for "complementary" polypurine and polypyrimidine tracks within 12 eukaryotic gene nucleotide sequences was carried out. Polypurine and polypyrimidine tracks (10-11 b.p.) potentially capable of interacting with each other with the formation of triplex structures ("structurizing" regions) has been shown to be located in chromosomal locus of genes, predominantly in introns and flanking regions. In the case of in vivo realization of such DNA-DNA interactions the chromosomal gene domains can be folded into several small loops. An involvement of DNA triplexes in the chromosomal gene loci compactization may be associated with the gene functioning. The analogous analysis carried out for nucleotide sequences of long (LINE) and short (SINE) repeats dispersed over the genome as well as of satellite DNA has demonstrated a fundamental identity between mechanisms of chromosomal encoding and non-coding regions' compactization.

  15. Atrophin-1全长基因稳定转染和瞬时转染真核细胞系的表达分析%Analysis of the expression of normal and mutant full-length atrophin-1 gene in stable and transient transfected eukaryotic cell line

    Institute of Scientific and Technical Information of China (English)

    张鑫; 顾卫红; 王国相

    2011-01-01

    Objective To establish eukaryotic expression system of full -length Sene in normal [CAG repeats (Q19)] and abnormal [CAG repeats (Q82 and Q66)] atrophin-1 [the pathogenic gene of dentatorubral-pallidoluysian atrophy (DRPLA)]. To compare the expression system of stable and transient transfected cell line by observing the cellular appearance and atrophin-1 fusion protein expression and its distribution for further research on the establishment of a fine cell model. Methods Based on the 2 established stable transfected green-fluoresent protein (GFP)-atrophin- 1-Q19/Flp- In TREx293 and GFP-atrophin - 1 -Q82/Flp -In TREx293 cell lines, Tel-on system was used to induce the expression, which contained normal and ahnormal atrophin - 1 full - length gene and GFP fusion proteins, respectively.Meanwhile, GFP - atrophin - 1 - Q19 and GFP - atrophin - 1 - Q66 recombinant plasmids were transiently transfected into 293T cells and SH-SY5Y cells respectively with LipofectamineTM 2000. The expression and localization of normal and abnormal atrophin - 1 fusion proteins in the cells were detected by light microscope with HE staining and fluorescence microscope; the ultrastructure of the cells were observed by electron microscope; the expression of GFP - atrophin - 1 fusion proteins were detected by Western blotting. Results In stable transfected system, the green fluorescence signals were detected in the nucleus, and abnormal proteins were not uniformly distributed while some were aggregated. Electron microscope showed shrinkaged nuclear membrane and abnormal intranuclear structure in abnormal cells. In transjpnt transfection of 293T, Q66 protein aggregated in the nucleus which was detected as acidophilic bodies by HE staining-light microscope and atrophin-1 fusion protein as punctuate pattern by fluorescence microscope. Under the observation of electron microscope, great quanlity of electron-dense substance accumulation, distinct shrinkage nuclear membrane and nucleus disappearance

  16. Sistema de detección y clasificación automática de granos de polen mediante técnicas de procesado digital de imágenes

    Directory of Open Access Journals (Sweden)

    Jorge Arroyo Hernández

    2013-03-01

    Full Text Available En este artículo se presentan los avances en la construcción de un sistema informático que permitirá el reconocimiento y clasificación taxonómica de granos de polen de algunas de las plantas melíferas tropicales más importantes en Costa Rica. Se aplicaron técnicas de pre y post procesado de imágenes digitales a partir de una base de datos de referencia. El sistema digital elaborado aplica filtros a las imágenes, lo cual permite su detección y un realce de sus características y su contorno. Luego, se parametriza y, finalmente, se utiliza un sistema de redes neuronales para el reconocimiento automático de los granos de polen. A través de la implementación de programas informáticos, se pretende pasar de un paradigma cualitativo a uno cuantitativo con el empleo de distintas herramientas matemáticas e inteligencia artificial, de forma que se pueda agilizar el proceso de reconocimiento y clasificación de los granos de polen. Mediante el método de PCA y la suma en las salidas de 30 redes neuronales (AS se logro obtener una tasa de éxito del 91,67± 3,13, lo cual es altamente promisorio para los efectos del sistema de clasificación automática.

  17. High genetic diversity and novelty in eukaryotic plankton assemblages inhabiting saline lakes in the Qaidam basin.

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    Jiali Wang

    Full Text Available Saline lakes are intriguing ecosystems harboring extremely productive microbial communities in spite of their extreme environmental conditions. We performed a comprehensive analysis of the genetic diversity (18S rRNA gene of the planktonic microbial eukaryotes (nano- and picoeukaryotes in six different inland saline lakes located in the Qaidam Basin. The novelty level are high, with about 11.23% of the whole dataset showing <90% identity to any previously reported sequence in GenBank. At least 4 operational taxonomic units (OTUs in mesosaline lakes, while up to eighteen OTUs in hypersaline lakes show very low CCM and CEM scores, indicating that these sequences are highly distantly related to any existing sequence. Most of the 18S rRNA gene sequence reads obtained in investigated mesosaline lakes is closely related to Holozoa group (48.13%, whereas Stramenopiles (26.65% and Alveolates (10.84% are the next most common groups. Hypersaline lakes in the Qaidam Basin are also dominated by Holozoa group, accounting for 26.65% of the total number of sequence reads. Notably, Chlorophyta group are only found in high abundance in Lake Gasikule (28.00%, whereas less represented in other hypersaline lakes such as Gahai (0.50% and Xiaochaidan (1.15%. Further analysis show that the compositions of planktonic eukaryotic assemblages are also most variable between different sampling sites in the same lake. Out of the parameters, four show significant correlation to this CCA: altitude, calcium, sodium and potassium concentrations. Overall, this study shows important gaps in the current knowledge about planktonic microbial eukaryotes inhabiting Qaidam Basin (hyper saline water bodies. The identified diversity and novelty patterns among eukaryotic plankton assemblages in saline lake are of great importance for understanding and interpreting their ecology and evolution.

  18. Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa.

    Science.gov (United States)

    Cavalier-Smith, Thomas; Chao, Ema E; Snell, Elizabeth A; Berney, Cédric; Fiore-Donno, Anna Maria; Lewis, Rhodri

    2014-12-01

    Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were previously argued to have evolved independently from the little-studied, largely flagellate, protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and the more primitive excavate flagellates that have ventral feeding grooves and the most primitive known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote-wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between homogeneous-model maximum-likelihood trees and evolutionarily more realistic site-heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 'podiate' clade, one of only three eukaryotic 'supergroups'. Our trees indicate that evolution of sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously

  19. A pipeline for automated annotation of yeast genome sequences by a conserved-synteny approach

    Directory of Open Access Journals (Sweden)

    Proux-Wéra Estelle

    2012-09-01

    Full Text Available Abstract Background Yeasts are a model system for exploring eukaryotic genome evolution. Next-generation sequencing technologies are poised to vastly increase the number of yeast genome sequences, both from resequencing projects (population studies and from de novo sequencing projects (new species. However, the annotation of genomes presents a major bottleneck for de novo projects, because it still relies on a process that is largely manual. Results Here we present the Yeast Genome Annotation Pipeline (YGAP, an automated system designed specifically for new yeast genome sequences lacking transcriptome data. YGAP does automatic de novo annotation, exploiting homology and synteny information from other yeast species stored in the Yeast Gene Order Browser (YGOB database. The basic premises underlying YGAP's approach are that data from other species already tells us what genes we should expect to find in any particular genomic region and that we should also expect that orthologous genes are likely to have similar intron/exon structures. Additionally, it is able to detect probable frameshift sequencing errors and can propose corrections for them. YGAP searches intelligently for introns, and detects tRNA genes and Ty-like elements. Conclusions In tests on Saccharomyces cerevisiae and on the genomes of Naumovozyma castellii and Tetrapisispora blattae newly sequenced with Roche-454 technology, YGAP outperformed another popular annotation program (AUGUSTUS. For S. cerevisiae and N. castellii, 91-93% of YGAP's predicted gene structures were identical to those in previous manually curated gene sets. YGAP has been implemented as a webserver with a user-friendly interface at http://wolfe.gen.tcd.ie/annotation.

  20. Cryptic sex in the smallest eukaryotic marine green alga.

    Science.gov (United States)

    Grimsley, Nigel; Péquin, Bérangère; Bachy, Charles; Moreau, Hervé; Piganeau, Gwenaël

    2010-01-01

    Ostreococcus spp. are common worldwide oceanic picoeukaryotic pelagic algae. The complete genomes of three strains from different ecological niches revealed them to represent biologically distinct species despite their identical cellular morphologies (cryptic species). Their tiny genomes (13 Mb), with approximately 20 chromosomes, are colinear and densely packed with coding sequences, but no sexual life cycle has been described. Seventeen new strains of one of these species, Ostreococcus tauri, were isolated from 98 seawater samplings from the NW Mediterranean by filtering, culturing, cloning, and plating for single colonies and identification by sequencing their ribosomal 18S gene. In order to find the genetic markers for detection of polymorphisms and sexual recombination, we used an in silico approach to screen available genomic data. Intergenic regions of DNA likely to evolve neutrally were analyzed following polymerase chain reaction amplification of sequences using flanking primers from adjacent conserved coding sequences that were present as syntenic pairs in two different species of Ostreococcus. Analyses of such DNA regions from eight marker loci on two chromosomes from each strain revealed that the isolated O. tauri clones were haploid and that the overall level of polymorphism was approximately 0.01. Four different genetic tests for recombination showed that sexual exchanges must be inferred to account for the between-locus and between-chromosome marker combinations observed. However, our data suggest that sexual encounters are infrequent because we estimate the frequency of meioses/mitoses among the sampled strains to be 10(-6). Ostreococcus tauri and related species encode and express core genes for mitosis and meiosis, but their mechanisms of cell division and recombination, nevertheless, remain enigmatic because a classical eukaryotic spindle with 40 canonical microtubules would be much too large for the available approximately 0.9-microm(3) cellular

  1. Diversity and dynamics of active microbial eukaryotes in the anoxic zone of a freshwater meromictic lake (Pavin, France

    Directory of Open Access Journals (Sweden)

    CECILE eLEPERE

    2016-02-01

    Full Text Available Microbial eukaryotes play a crucial role in ecosystem functioning and oxygen is considered to be one of the strongest barriers against their local dispersal. However, diversity of microbial eukaryotes in freshwater habitats with oxygen gradients has previously received very little attention. We applied high-throughput sequencing (V4 region of the 18S rRNA gene in conjunction with quantitative PCR (DNA and RNA and fluorescent in situ hybridization analyses, to provide an unique spatio-temporal analysis of microbial eukaryotes diversity and potential activity in a meromictic freshwater lake (lake Pavin. This study revealed a high genetic diversity of unicellular eukaryotes in the permanent anoxic zone of lake Pavin and allowed the discrimination of active vs. inactive components. 42 % of the OTUs (Operational taxonomic Units are exclusively present in the monimolimnion, where Alveolata (Ciliophora and Dinophyceae and Fungi (Dikarya and Chytrids are the most active phyla and are probably represented by species capable of anaerobic metabolism. Pigmented eukaryotes (Haptophyceae and Chlorophyceae are also present and active in this zone, which opens up questions regarding their metabolism.

  2. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

    Directory of Open Access Journals (Sweden)

    Santiago Guerrero

    2015-01-01

    Full Text Available Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1 uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication.

  3. Construction of Eukaryotic Co-expression Plasmid Carrying pGH and IGF-Ⅰ Gene and It′s Transformation in Landrace%pGH 和 IGF-Ⅰ双基因共表达载体的构建及转双基因猪的获得与检测

    Institute of Scientific and Technical Information of China (English)

    姚延珠; 吴明明; 孙金海

    2016-01-01

    cultivation of new breed of feed-saving grain pigs.Total RNA was extracted from the ear tissue of Landrace pig,and the coding sequence of pGH without termination codon and complete coding sequence of IGF-Ⅰ were amplified by RT-PCR and cloned into the pcDNA3.1 (+)eukaryotic expression vector after verified by sequencing.The recombinant plasmid was verified by sequencing and enzyme digestion and then transfected into PK15 cells.Expression of the two genes in PK15 cells was detected by Q-PCR.Transgenic pigs were prepared with sperm-mediated transformation after the sperm were encapsulated by nanometer material.Transgenic pigs were iden-tified by PCR and sequencing.The expression of the two target genes were detected by Q-PCR.Stability of transgene was detected by PCR and sequencing aged from 1 month to 7 month.RT-PCR and sequencing results showed that the coding sequences of pGH and IGF-Ⅰ of Landrace were successfully cloned.Digestion and sequencing analysis showed the eukaryotic co-expression vector containing pGH and IGF-Ⅰ was successfully constructed,and Q-PCR a-nalysis showed that pGH and IGF-Ⅰ were successfully expressed at the mRNA level after transfected into PK15 cells.As a result,13 young piglets were born,and 4 of them were positive for both two genes detected by PCR and sequencing.As a result,the positive rate was 30.76%.Q-PCR results showed that exogenous pGH and IGF-Ⅰ were successful expressed in transgenic pigs.Exogenous pGH and IGF-Ⅰ could be detected in transgenic positive individ-uals from aged 1 month to 7 month,which proved the two exogenous genes were stable in transgenic pigs and were not lost in growth process.Exogenous pGH and IGF-Ⅰ could be detected in sperm of transgenic boars showed that exogenous pGH and IGF-Ⅰ may be passaged stable.

  4. Automated DNA Sequencing System

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  5. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  6. Membranes, energetics, and evolution across the prokaryote-eukaryote divide

    Science.gov (United States)

    Lynch, Michael; Marinov, Georgi K

    2017-01-01

    The evolution of the eukaryotic cell marked a profound moment in Earth’s history, with most of the visible biota coming to rely on intracellular membrane-bound organelles. It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells. However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes. Thus, although the origin of the mitochondrion was a key event in evolutionary history, there is no reason to think membrane bioenergetics played a direct, causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity. DOI: http://dx.doi.org/10.7554/eLife.20437.001 PMID:28300533

  7. The structure and function of the eukaryotic ribosome.

    Science.gov (United States)

    Wilson, Daniel N; Doudna Cate, Jamie H

    2012-05-01

    Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented.

  8. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

    NARCIS (Netherlands)

    Wijffels, R.H.; Kruse, O.; Hellingwerf, K.J.

    2013-01-01

    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 fo

  9. A statistical anomaly indicates symbiotic origins of eukaryotic membranes.

    Science.gov (United States)

    Bansal, Suneyna; Mittal, Aditya

    2015-04-01

    Compositional analyses of nucleic acids and proteins have shed light on possible origins of living cells. In this work, rigorous compositional analyses of ∼5000 plasma membrane lipid constituents of 273 species in the three life domains (archaea, eubacteria, and eukaryotes) revealed a remarkable statistical paradox, indicating symbiotic origins of eukaryotic cells involving eubacteria. For lipids common to plasma membranes of the three domains, the number of carbon atoms in eubacteria was found to be similar to that in eukaryotes. However, mutually exclusive subsets of same data show exactly the opposite-the number of carbon atoms in lipids of eukaryotes was higher than in eubacteria. This statistical paradox, called Simpson's paradox, was absent for lipids in archaea and for lipids not common to plasma membranes of the three domains. This indicates the presence of interaction(s) and/or association(s) in lipids forming plasma membranes of eubacteria and eukaryotes but not for those in archaea. Further inspection of membrane lipid structures affecting physicochemical properties of plasma membranes provides the first evidence (to our knowledge) on the symbiotic origins of eukaryotic cells based on the "third front" (i.e., lipids) in addition to the growing compositional data from nucleic acids and proteins.

  10. An Evolutionary Framework for Understanding the Origin of Eukaryotes.

    Science.gov (United States)

    Blackstone, Neil W

    2016-04-27

    Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real-the endosymbiosis that led to the mitochondrion is often described as "non-Darwinian" because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious-all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

  11. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  12. On the Diversification of the Translation Apparatus across Eukaryotes

    Directory of Open Access Journals (Sweden)

    Greco Hernández

    2012-01-01

    Full Text Available Diversity is one of the most remarkable features of living organisms. Current assessments of eukaryote biodiversity reaches 1.5 million species, but the true figure could be several times that number. Diversity is ingrained in all stages and echelons of life, namely, the occupancy of ecological niches, behavioral patterns, body plans and organismal complexity, as well as metabolic needs and genetics. In this review, we will discuss that diversity also exists in a key biochemical process, translation, across eukaryotes. Translation is a fundamental process for all forms of life, and the basic components and mechanisms of translation in eukaryotes have been largely established upon the study of traditional, so-called model organisms. By using modern genome-wide, high-throughput technologies, recent studies of many nonmodel eukaryotes have unveiled a surprising diversity in the configuration of the translation apparatus across eukaryotes, showing that this apparatus is far from being evolutionarily static. For some of the components of this machinery, functional differences between different species have also been found. The recent research reviewed in this article highlights the molecular and functional diversification the translational machinery has undergone during eukaryotic evolution. A better understanding of all aspects of organismal diversity is key to a more profound knowledge of life.

  13. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    Science.gov (United States)

    Blackstone, Neil W.

    2016-01-01

    Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real—the endosymbiosis that led to the mitochondrion is often described as “non-Darwinian” because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious—all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes. PMID:27128953

  14. Crystal structure of eukaryotic ribosome and its complexes with inhibitors.

    Science.gov (United States)

    Yusupova, Gulnara; Yusupov, Marat

    2017-03-19

    A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome-the high-eukaryote-specific long ribosomal RNA segments (about 1MDa)-remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved.This article is part of the themed issue 'Perspectives on the ribosome'.

  15. An Evolutionary Framework for Understanding the Origin of Eukaryotes

    Directory of Open Access Journals (Sweden)

    Neil W. Blackstone

    2016-04-01

    Full Text Available Two major obstacles hinder the application of evolutionary theory to the origin of eukaryotes. The first is more apparent than real—the endosymbiosis that led to the mitochondrion is often described as “non-Darwinian” because it deviates from the incremental evolution championed by the modern synthesis. Nevertheless, endosymbiosis can be accommodated by a multi-level generalization of evolutionary theory, which Darwin himself pioneered. The second obstacle is more serious—all of the major features of eukaryotes were likely present in the last eukaryotic common ancestor thus rendering comparative methods ineffective. In addition to a multi-level theory, the development of rigorous, sequence-based phylogenetic and comparative methods represents the greatest achievement of modern evolutionary theory. Nevertheless, the rapid evolution of major features in the eukaryotic stem group requires the consideration of an alternative framework. Such a framework, based on the contingent nature of these evolutionary events, is developed and illustrated with three examples: the putative intron proliferation leading to the nucleus and the cell cycle; conflict and cooperation in the origin of eukaryotic bioenergetics; and the inter-relationship between aerobic metabolism, sterol synthesis, membranes, and sex. The modern synthesis thus provides sufficient scope to develop an evolutionary framework to understand the origin of eukaryotes.

  16. Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity

    Science.gov (United States)

    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.

  17. Spy: a new group of eukaryotic DNA transposons without target site duplications.

    Science.gov (United States)

    Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

    2014-06-24

    Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties.

  18. Dramatic shifts in benthic microbial eukaryote communities following the Deepwater Horizon oil spill.

    Directory of Open Access Journals (Sweden)

    Holly M Bik

    Full Text Available Benthic habitats harbour a significant (yet unexplored diversity of microscopic eukaryote taxa, including metazoan phyla, protists, algae and fungi. These groups are thought to underpin ecosystem functioning across diverse marine environments. Coastal marine habitats in the Gulf of Mexico experienced visible, heavy impacts following the Deepwater Horizon oil spill in 2010, yet our scant knowledge of prior eukaryotic biodiversity has precluded a thorough assessment of this disturbance. Using a marker gene and morphological approach, we present an intensive evaluation of microbial eukaryote communities prior to and following oiling around heavily impacted shorelines. Our results show significant changes in community structure, with pre-spill assemblages of diverse Metazoa giving way to dominant fungal communities in post-spill sediments. Post-spill fungal taxa exhibit low richness and are characterized by an abundance of known hydrocarbon-degrading genera, compared to prior communities that contained smaller and more diverse fungal assemblages. Comparative taxonomic data from nematodes further suggests drastic impacts; while pre-spill samples exhibit high richness and evenness of genera, post-spill communities contain mainly predatory and scavenger taxa alongside an abundance of juveniles. Based on this community analysis, our data suggest considerable (hidden initial impacts across Gulf beaches may be ongoing, despite the disappearance of visible surface oil in the region.

  19. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.

    Science.gov (United States)

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; van Voorhis, Wesley C; Hol, Wim G J

    2010-03-26

    Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

  20. Rapid detection of Gram-negative bacteria and their drug resistance genes from positive blood cultures using an automated microarray assay.

    Science.gov (United States)

    Han, Eunhee; Park, Dong-Jin; Kim, Yukyoung; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

    2015-03-01

    We evaluated the performance of the Verigene Gram-negative blood culture (BC-GN) assay (CE-IVD version) for identification of Gram-negative (GN) bacteria and detection of resistance genes. A total of 163 GN organisms (72 characterized strains and 91 clinical isolates from 86 patients) were tested; among the clinical isolates, 86 (94.5%) isolates were included in the BC-GN panel. For identification, the agreement was 98.6% (146/148, 95% confidence interval [CI], 92.1-100) and 70% (7/10, 95% CI, 53.5-100) for monomicrobial and polymicrobial cultures, respectively. Of the 48 resistance genes harbored by 43 characterized strains, all were correctly detected. Of the 19 clinical isolates harboring resistance genes, 1 CTX-M-producing Escherichia coli isolated in polymicrobial culture was not detected. Overall, BC-GN assay provides acceptable accuracy for rapid identification of Gram-negative bacteria and detection of resistance genes, compared with routine laboratory methods despite that it has limitations in the number of genus/species and resistance gene included in the panel and it shows lower sensitivity in polymicrobial cultures.

  1. Laboratory Automation and Middleware.

    Science.gov (United States)

    Riben, Michael

    2015-06-01

    The practice of surgical pathology is under constant pressure to deliver the highest quality of service, reduce errors, increase throughput, and decrease turnaround time while at the same time dealing with an aging workforce, increasing financial constraints, and economic uncertainty. Although not able to implement total laboratory automation, great progress continues to be made in workstation automation in all areas of the pathology laboratory. This report highlights the benefits and challenges of pathology automation, reviews middleware and its use to facilitate automation, and reviews the progress so far in the anatomic pathology laboratory.

  2. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

    Science.gov (United States)

    Zhang, Mengmeng; Gill, Gordon N; Zhang, Yan

    2010-01-01

    In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  3. Characterization of SNAREs Determines the Absence of a Typical Golgi Apparatus in the Ancient Eukaryote Giardia lamblia*S⃞

    Science.gov (United States)

    Elias, Eliana V.; Quiroga, Rodrigo; Gottig, Natalia; Nakanishi, Hideki; Nash, Theodore E.; Neiman, Aaron; Lujan, Hugo D.

    2008-01-01

    Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen. PMID:18930915

  4. Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR.

    Science.gov (United States)

    Chiefari, Amy K; Perry, Michael J; Kelly-Cirino, Cassandra; Egan, Christina T

    2015-12-01

    To minimize specimen volume, handling and testing time, we have developed two TaqMan(®) multiplex real-time PCR (rtPCR) assays to detect staphylococcal enterotoxins A-E and Toxic Shock Syndrome Toxin production genes directly from clinical patient stool specimens utilizing a novel lysis extraction process in parallel with the Roche MagNA Pure Compact. These assays are specific, sensitive and reliable for the detection of the staphylococcal enterotoxin encoding genes and the tst1 gene from known toxin producing strains of Staphylococcus aureus. Specificity was determined by testing a total of 47 microorganism strains, including 8 previously characterized staphylococcal enterotoxin producing strains against each rtPCR target. Sensitivity for these assays range from 1 to 25 cfu per rtPCR reaction for cultured isolates and 8-20 cfu per rtPCR for the clinical stool matrix.

  5. Phylogenetic analysis of eukaryotes using heat-shock protein Hsp90.

    Science.gov (United States)

    Stechmann, Alexandra; Cavalier-Smith, Thomas

    2003-10-01

    Most eukaryote molecular phylogenies have been based on small-subunit ribosomal RNA as its database includes the most species, but serious problems have been encountered that can make these trees misleading. More recent studies using concatenated protein sequences have increased the data per organism, reducing misleading signals from a single sequence, but taxon sampling is limited. To increase the database of protein-coding genes we sequenced the cytosolic form of heat-shock protein Hsp90 from a broad variety of previously unsampled eukaryote groups: protozoan flagellates (phyla Choanozoa, Apusozoa, Cercozoa) and all three groups of chromists (Cryptophyta, Heterokonta, Haptophyta). Gamma-corrected distance trees robustly show three groups: bacterial sequences are sister to all eukaryote sequences, which are cleanly subdivided into the cytosolic sequences and a clade comprising the chloroplast and endoplasmic reticulum (ER) Hsp90 sequences. The eukaryote cytosolic sequences comprise a robust opisthokont clade (animals/Choanozoa/fungi), a bikont clade, and an amoebozoan branch. However their topology is not robust. When the cytosolic sequences are rooted using only the ER/ chloroplast clade as outgroup the amoebozoan Dictyostelium is sister to the opisthokonts forming a unikont clade in the distance tree. Congruence of this tree with that for concatenated mitochondrial proteins suggests that the root of the eukaryote tree is between unikonts and bikonts. Gamma-corrected maximum likelihood analyses of cytosolic sequences alone (519 unambiguously aligned amino acid positions) show bikonts as a clade, as do least-squares distance trees, but with other distance methods and parsimony the sole amoebozoan species branches weakly within bikonts. Choanozoa are clearly sisters to animals. Some major bikont groups (e.g. green plants, alveolates, Euglenozoa) are consistently recovered, but others (e.g. discicristates, chromalveolates) appear only in some trees; the backbone of

  6. Molecular phylogeny of centrohelid heliozoa, a novel lineage of bikont eukaryotes that arose by ciliary loss.

    Science.gov (United States)

    Cavalier-Smith, Thomas; Chao, Ema E-Y

    2003-04-01

    Recent molecular and cellular evidence indicates that eukaryotes comprise three major lineages: the probably ancestrally uniciliate protozoan phylum Amoebozoa; the ancestrally posteriorly uniciliate opisthokont clade (animals, Choanozoa, and fungi); and a very diverse ancestrally biciliate clade, the bikonts-plants, chromalveolates, and excavate and rhizarian Protozoa. As Heliozoa are the only eukaryote phylum not yet placed on molecular sequence trees, we have sequenced the 18S rRNA genes of three centrohelid heliozoa, Raphidiophrys ambigua, Heterophrys marina, and Chlamydaster sterni, to investigate their phylogenetic position. Phylogenetic analysis by distance and maximum likelihood methods allowing for intersite rate variation and invariable sites confirms that centrohelid heliozoa are a robust clade that does not fall within any other phyla. In particular, they are decisively very distant from the heterokont pedinellid chromists, at one time thought to be related to heliozoa, and lack the unique heterokont signature sequence. They also appear not to be specifically related to either Amoebozoa or Radiolaria, with which they have sometimes been classified, so it is desirable to retain Heliozoa as a separate protozoan phylum. Even though centrohelids have no cilia or centrioles, the centrohelid clade branches among the bikont eukaryotes, but there is no strong bootstrap support for any particular position. Distance trees usually place centrohelids as sisters to haptophytes, whereas parsimony puts them as sisters to red algae, but there is no reason to think that either position is correct; both have very low bootstrap support. Quartet puzzling places them with fairly low support as sisters to the apusozoan zooflagellate Ancyromonas. As Ancyromonas is the only other eukaryote that shares the character combination of flat plate-like mitochondrial cristae and kinetocyst-type extrusomes with centrohelids, this position is biologically plausible, but because of weak

  7. Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean.

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

    Full Text Available BACKGROUND: Photosynthetic picoeukaryotes (PPE with a cell size less than 3 microm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone. METHODOLOGY/PRINCIPAL FINDINGS: In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX, clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales dominated. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics

  8. Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae).

    Science.gov (United States)

    Cavalier-Smith, T

    2003-01-29

    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

  9. Construction of shRNA eukaryotic expression vectors targeting PERK gene and effect of PERK gene knockdown on apoptosis in endoplasmic reticulum stress-induced L02 hepatocytes%靶向PERK基因的shRNA真核表达载体的构建及对内质网应激状态下L02肝细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    曹洁; 杨朝霞; 沈薇; 姚隆

    2011-01-01

    AIM: To construct short hairpin RNA ( shRNA ) eukaryotic expression vectors targeting the gene of RNA - dependent protein kinase ( PKR ) - like endoplasmic reticulum kinase ( PERK ), and to observe the effect of PERK gene knockdown on apoptosis of human normal hepatic L02 cells treated with thapsigargin. METHODS: Three shRNA expression vectors targeting PERK gene, named PERK1 - shRNA, PERK2 - shRNA and PERKS - shRNA, and one non -homologous negative control expression vector ( HK - shRNA ) were constructed based on the nucleotide sequence of PERK and the criteria of designing small interfering RNA ( siRNA ), and were identified by enzyme digestion and DNA sequencing analysis. After L02 hepatocytes were transfected with the plasmids, the PERK expression was determined by RT - PCR and Western blotting, and the plasmid with the best inhibitory effect on PERK expression was screened. The cell viability and apoptotic rate of L02 hepatocytes transfected with PERK - shRNA under endoplasmic reticulum stress ( ERS ) were measured by the methods of MTT and flow cytometry,respectively. RESULTS: Four shRNA expression vectors were constructed. The mRNA and protein expression levels of PERK gene decreased significantly in L02 hepatocytes transfected with PERK1 - shRNA, PERK2 - shRNA and PERK3 - shRNA as compared with those in control cells ( P <0. 05 ). The inter-fering effect of PERK1 - shRNA on PERK gene expression was the best. PERK knockdown by PERK1 - shRNA increased the viability and inhibited the apoptosis of L02 cells under ERS. CONCLUSION: The shRNA expression vectors targeting PERK gene are constructed, and PERK gene knockdown may inhibit apoptosis in L02 hepatocytes under ERS.%目的:构建靶向RNA依赖的蛋白激酶样内质网激酶(PERK)基因的短发夹状RNA(shRNA)真核表达载体,观察其对内质网应激(ERS)状态下人正常肝细胞株L02凋亡的影响.方法:根据PERK基因核苷酸序列,按照小干扰RNA(siRNA)靶序列的设计原则,设

  10. Sequence Similarity and Functional Relationship Among Eukaryotic ZIP and CDF Transporters

    Institute of Scientific and Technical Information of China (English)

    Taiho Kambe; Tomoyuki Suzuki; Masaya Nagao; Yuko Yamaguchi-Iwai

    2006-01-01

    ZIP (ZRT/IRT-like Protein) and CDF (Cation Diffusion Facilitator) are two large metal transporter families mainly transporting zinc into and out of the cytosol.Several ZIP and CDF transporters have been characterized in mammals and various model organisms, such as yeast, nematode, fruit fly, and zebrafish, and many candidate genes have been identified by genome projects. Unexpected functions of ZIP and CDF transporters have been recently reported in some model organisms,leading to major advances in our understanding of the functions of mammalian counterparts. Here, we review the recent information on the sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters obtained from the representative model organisms.

  11. Basal transcription machinery: role in regulation of stress response in eukaryotes

    Indian Academy of Sciences (India)

    Parag Sadhale; Jiyoti Verma; Aruna Naorem

    2007-04-01

    The holoenzyme of prokaryotic RNA polymerase consists of the core enzyme, made of two , , ’ and subunits, which lacks promoter selectivity and a sigma () subunit which enables the core enzyme to initiate transcription in a promoter dependent fashion. A stress sigma factor s, in prokaryotes seems to regulate several stress response genes in conjunction with other stress specific regulators. Since the basic principles of transcription are conserved from simple bacteria to multicellular complex organisms, an obvious question is: what is the identity of a counterpart of s, that is closest to the core polymerase and that dictates transcription of stress regulated genes in general? In this review, we discuss the logic behind the suggestion that like in prokaryotes, eukaryotes also have a common functional unit in the transcription machinery through which the stress specific transcription factors regulate rapid and highly controlled induction of gene expression associated with generalized stress response and point to some candidates that would fit the bill of the eukaryotic s.

  12. Distinct regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID.

    Science.gov (United States)

    Bhaumik, Sukesh R

    2011-02-01

    A growing number of human diseases are linked to abnormal gene expression which is largely controlled at the level of transcriptional initiation. The gene-specific activator promotes the initiation of transcription through its interaction with one or more components of the transcriptional initiation machinery, hence leading to stimulated transcriptional initiation or activation. However, all activator proteins do not target the same component(s) of the transcriptional initiation machinery. Rather, they can have different target specificities, and thus, can lead to distinct mechanisms of transcriptional activation. Two such distinct mechanisms of transcriptional activation in yeast are mediated by the SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (Transcription factor IID) complexes, and are termed as "SAGA-dependent" and "TFIID-dependent" transcriptional activation, respectively. SAGA is the target of the activator in case of SAGA-dependent transcriptional activation, while the targeting of TFIID by the activator leads to TFIID-dependent transcriptional activation. Both the SAGA and TFIID complexes are highly conserved from yeast to human, and play crucial roles in gene activation among eukaryotes. The regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID are discussed here. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

  13. Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

    Directory of Open Access Journals (Sweden)

    Nicola Vitulo

    2007-01-01

    Full Text Available The MOB family includes a group of cell cycle-associated proteins highly conserved throughout eukaryotes, whose founding members are implicated in mitotic exit and co-ordination of cell cycle progression with cell polarity and morphogenesis. Here we report the characterization and evolution of the MOB domain-containing proteins as inferred from the 43 eukaryotic genomes so far sequenced. We show that genes for Mob-like proteins are present in at least 41 of these genomes, confi rming the universal distribution of this protein family and suggesting its prominent biological function. The phylogenetic analysis reveals fi ve distinct MOB domain classes, showing a progressive expansion of this family from unicellular to multicellular organisms, reaching the highest number in mammals. Plant Mob genes appear to have evolved from a single ancestor, most likely after the loss of one or more genes during the early stage of Viridiplantae evolutionary history. Three of the Mob classes are widespread among most of the analyzed organisms. The possible biological and molecular function of Mob proteins and their role in conserved signaling pathways related to cell proliferation, cell death and cell polarity are also presented and critically discussed.

  14. Cloning, Expression and Function Analyses of Eukaryotic Translation Initiation Factor 4E(BmeIF4E) Gene from Silkworm Pupa (Bombyx mori)%家蚕真核细胞翻译起始因子(BmeIF4E)基因的克隆、表达及功能分析

    Institute of Scientific and Technical Information of China (English)

    于威; 韩剑秋; 张耀洲

    2011-01-01

    The full open reading frame (ORF) of eukaryotic translation initiation factor 4E (eIF4E) was obtained from silkworm(Bombyx mori pupal cDNA library and was named BmeIF4E (GenBank accession No.DN443192).In order to study the biological function of BmeIF4E, the ORF of this gene was cloned and the procaryotic expression recombinant plasmid pET-28a(+)-BmeIF4E was constructed to express His-BmeIF4E fusion protein in Escherichia coli BL21 (DE3).Because the rBmeIF4E in E.coli was soluble, the protein was purified with metal-chelating affinity chromatography directly, rBmeIF4E was used to generate anti-BmeIF4E polyclonal antibodies, to determine the subcellular localization of BmeIF4E.Immnunostaining indicated that BmeIF4E protein could be found in both the cytoplasm and nucleus.The gene expression features in different organizations and different developmental stages were analyzed using Real-time RT-PCR and Western blotting analyses.The results showed that BmeIF4E were expressed in all tissues and the expression levels were highest in malpighian tubes,followed by epidermis, fat body, spiracle, ovary, gut and head, and were lowest in the silk glad.Additionally,BmeIF4E expression began during the egg stage, increased during the larvae stage and pupa stage, reached a peak during the moth stage.Furthermore, RNA interference technology was applied to silence the target mRNA and the cell viability was assessed by Western blot and MTT assay.After 72 h of RNAi, the MTT assay indicated that when BmeIF4E was knocked down in Bm5 cells, the cell viability was decreased significantly.BmeIF4E may play an important role in the whole life cycle ofBombyx mori as a valuable house-keeping gene.%本研究从自建的家蚕(Bombyx mori)蛹期cDNA文库中筛选到一条cDNA序列,发现其在序列和结构上与其他物种的真核细胞翻译起始因子钮基因(BmeIF4E)具有较高的相似性,推测可能是家蚕eIF4E基因,将该序列命名为BmeIF4E,GenBank登录号为DN443192.

  15. Predation and eukaryote cell origins: a coevolutionary perspective.

    Science.gov (United States)

    Cavalier-Smith, T

    2009-02-01

    Cells are of only two kinds: bacteria, with DNA segregated by surface membrane motors, dating back approximately 3.5Gy; and eukaryotes, which evolved from bacteria, possibly as recently as 800-850My ago. The last common ancestor of eukaryotes was a sexual phagotrophic protozoan with mitochondria, one or two centrioles and cilia. Conversion of bacteria (=prokaryotes) into a eukaryote involved approximately 60 major innovations. Numerous contradictory ideas about eukaryogenesis fail to explain fundamental features of eukaryotic cell biology or conflict with phylogeny. Data are best explained by the intracellular coevolutionary theory, with three basic tenets: (1) the eukaryotic cytoskeleton and endomembrane system originated through cooperatively enabling the evolution of phagotrophy; (2) phagocytosis internalised DNA-membrane attachments, unavoidably disrupting bacterial division; recovery entailed the evolution of the nucleus and mitotic cycle; (3) the symbiogenetic origin of mitochondria immediately followed the perfection of phagotrophy and intracellular digestion, contributing greater energy efficiency and group II introns as precursors of spliceosomal introns. Eukaryotes plus their archaebacterial sisters form the clade neomura, which evolved from a radically modified derivative of an actinobacterial posibacterium that had replaced the ancestral eubacterial murein peptidoglycan by N-linked glycoproteins, radically modified its DNA-handling enzymes, and evolved cotranslational protein secretion, but not the isoprenoid-ether lipids of archaebacteria. I focus on this phylogenetic background and on explaining how in response to novel phagotrophic selective pressures and ensuing genome internalisation this prekaryote evolved efficient digestion of prey proteins by retrotranslocation and 26S proteasomes, then internal digestion by phagocytosis, lysosomes, and peroxisomes, and eukaryotic vesicle trafficking and intracellular compartmentation.

  16. Automating checks of plan check automation.

    Science.gov (United States)

    Halabi, Tarek; Lu, Hsiao-Ming

    2014-07-08

    While a few physicists have designed new plan check automation solutions for their clinics, fewer, if any, managed to adapt existing solutions. As complex and varied as the systems they check, these programs must gain the full confidence of those who would run them on countless patient plans. The present automation effort, planCheck, therefore focuses on versatility and ease of implementation and verification. To demonstrate this, we apply planCheck to proton gantry, stereotactic proton gantry, stereotactic proton fixed beam (STAR), and IMRT treatments.

  17. Automation in Warehouse Development

    NARCIS (Netherlands)

    Hamberg, R.; Verriet, J.

    2012-01-01

    The warehouses of the future will come in a variety of forms, but with a few common ingredients. Firstly, human operational handling of items in warehouses is increasingly being replaced by automated item handling. Extended warehouse automation counteracts the scarcity of human operators and support

  18. Automate functional testing

    Directory of Open Access Journals (Sweden)

    Ramesh Kalindri

    2014-06-01

    Full Text Available Currently, software engineers are increasingly turning to the option of automating functional tests, but not always have successful in this endeavor. Reasons range from low planning until over cost in the process. Some principles that can guide teams in automating these tests are described in this article.

  19. More Benefits of Automation.

    Science.gov (United States)

    Getz, Malcolm

    1988-01-01

    Describes a study that measured the benefits of an automated catalog and automated circulation system from the library user's point of view in terms of the value of time saved. Topics discussed include patterns of use, access time, availability of information, search behaviors, and the effectiveness of the measures used. (seven references)…

  20. Advances in inspection automation

    Science.gov (United States)

    Weber, Walter H.; Mair, H. Douglas; Jansen, Dion; Lombardi, Luciano

    2013-01-01

    This new session at QNDE reflects the growing interest in inspection automation. Our paper describes a newly developed platform that makes the complex NDE automation possible without the need for software programmers. Inspection tasks that are tedious, error-prone or impossible for humans to perform can now be automated using a form of drag and drop visual scripting. Our work attempts to rectify the problem that NDE is not keeping pace with the rest of factory automation. Outside of NDE, robots routinely and autonomously machine parts, assemble components, weld structures and report progress to corporate databases. By contrast, components arriving in the NDT department typically require manual part handling, calibrations and analysis. The automation examples in this paper cover the development of robotic thickness gauging and the use of adaptive contour following on the NRU reactor inspection at Chalk River.

  1. Automation in immunohematology.

    Science.gov (United States)

    Bajpai, Meenu; Kaur, Ravneet; Gupta, Ekta

    2012-07-01

    There have been rapid technological advances in blood banking in South Asian region over the past decade with an increasing emphasis on quality and safety of blood products. The conventional test tube technique has given way to newer techniques such as column agglutination technique, solid phase red cell adherence assay, and erythrocyte-magnetized technique. These new technologies are adaptable to automation and major manufacturers in this field have come up with semi and fully automated equipments for immunohematology tests in the blood bank. Automation improves the objectivity and reproducibility of tests. It reduces human errors in patient identification and transcription errors. Documentation and traceability of tests, reagents and processes and archiving of results is another major advantage of automation. Shifting from manual methods to automation is a major undertaking for any transfusion service to provide quality patient care with lesser turnaround time for their ever increasing workload. This article discusses the various issues involved in the process.

  2. Automated model building

    CERN Document Server

    Caferra, Ricardo; Peltier, Nicholas

    2004-01-01

    This is the first book on automated model building, a discipline of automated deduction that is of growing importance Although models and their construction are important per se, automated model building has appeared as a natural enrichment of automated deduction, especially in the attempt to capture the human way of reasoning The book provides an historical overview of the field of automated deduction, and presents the foundations of different existing approaches to model construction, in particular those developed by the authors Finite and infinite model building techniques are presented The main emphasis is on calculi-based methods, and relevant practical results are provided The book is of interest to researchers and graduate students in computer science, computational logic and artificial intelligence It can also be used as a textbook in advanced undergraduate courses

  3. Automation in Warehouse Development

    CERN Document Server

    Verriet, Jacques

    2012-01-01

    The warehouses of the future will come in a variety of forms, but with a few common ingredients. Firstly, human operational handling of items in warehouses is increasingly being replaced by automated item handling. Extended warehouse automation counteracts the scarcity of human operators and supports the quality of picking processes. Secondly, the development of models to simulate and analyse warehouse designs and their components facilitates the challenging task of developing warehouses that take into account each customer’s individual requirements and logistic processes. Automation in Warehouse Development addresses both types of automation from the innovative perspective of applied science. In particular, it describes the outcomes of the Falcon project, a joint endeavour by a consortium of industrial and academic partners. The results include a model-based approach to automate warehouse control design, analysis models for warehouse design, concepts for robotic item handling and computer vision, and auton...

  4. Automation in Immunohematology

    Directory of Open Access Journals (Sweden)

    Meenu Bajpai

    2012-01-01

    Full Text Available There have been rapid technological advances in blood banking in South Asian region over the past decade with an increasing emphasis on quality and safety of blood products. The conventional test tube technique has given way to newer techniques such as column agglutination technique, solid phase red cell adherence assay, and erythrocyte-magnetized technique. These new technologies are adaptable to automation and major manufacturers in this field have come up with semi and fully automated equipments for immunohematology tests in the blood bank. Automation improves the objectivity and reproducibility of tests. It reduces human errors in patient identification and transcription errors. Documentation and traceability of tests, reagents and processes and archiving of results is another major advantage of automation. Shifting from manual methods to automation is a major undertaking for any transfusion service to provide quality patient care with lesser turnaround time for their ever increasing workload. This article discusses the various issues involved in the process.

  5. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

    Science.gov (United States)

    Denks, Kärt; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

  6. Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs with an emphasis on poplar

    Directory of Open Access Journals (Sweden)

    Duplessis Sébastien

    2011-02-01

    Full Text Available Abstract Background Plant inducible immunity includes the accumulation of a set of defense proteins during infection called pathogenesis-related (PR proteins, which are grouped into families termed PR-1 to PR-17. The PR-5 family is composed of thaumatin-like proteins (TLPs, which are responsive to biotic and abiotic stress and are widely studied in plants. TLPs were also recently discovered in fungi and animals. In the poplar genome, TLPs are over-represented compared with annual species and their transcripts strongly accumulate during stress conditions. Results Our analysis of the poplar TLP family suggests that the expansion of this gene family was followed by diversification, as differences in expression patterns and predicted properties correlate with phylogeny. In particular, we identified a clade of poplar TLPs that cluster to a single 350 kb locus of chromosome I and that are up-regulated by poplar leaf rust infection. A wider phylogenetic analysis of eukaryote TLPs - including plant, animal and fungi sequences - shows that TLP gene content and diversity increased markedly during land plant evolution. Mapping the reported functions of characterized TLPs to the eukaryote phylogenetic tree showed that antifungal or glycan-lytic properties are widespread across eukaryote phylogeny, suggesting that these properties are shared by most TLPs and are likely associated with the presence of a conserved acidic cleft in their 3D structure. Also, we established an exhaustive catalog of TLPs with atypical architectures such as small-TLPs, TLP-kinases and small-TLP-kinases, which have potentially developed alternative functions (such as putative receptor kinases for pathogen sensing and signaling. Conclusion Our study, based on the most recent plant genome sequences, provides evidence for TLP gene family diversification during land plant evolution. We have shown that the diverse functions described for TLPs are not restricted to specific clades but seem

  7. Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.

    Directory of Open Access Journals (Sweden)

    Dale N Richardson

    Full Text Available Alternative splicing (AS of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes. AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms.

  8. Nitrate storage and dissimilatory nitrate reduction by eukaryotic microbes

    DEFF Research Database (Denmark)

    Kamp, Anja; Høgslund, Signe; Risgaard-Petersen, Nils;

    2015-01-01

    The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly...... and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate...... storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described...

  9. Interaction of tRNA with Eukaryotic Ribosome

    Directory of Open Access Journals (Sweden)

    Dmitri Graifer

    2015-03-01

    Full Text Available This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria.

  10. Systematic review automation technologies

    Science.gov (United States)

    2014-01-01

    Systematic reviews, a cornerstone of evidence-based medicine, are not produced quickly enough to support clinical practice. The cost of production, availability of the requisite expertise and timeliness are often quoted as major contributors for the delay. This detailed survey of the state of the art of information systems designed to support or automate individual tasks in the systematic review, and in particular systematic reviews of randomized controlled clinical trials, reveals trends that see the convergence of several parallel research projects. We surveyed literature describing informatics systems that support or automate the processes of systematic review or each of the tasks of the systematic review. Several projects focus on automating, simplifying and/or streamlining specific tasks of the systematic review. Some tasks are already fully automated while others are still largely manual. In this review, we describe each task and the effect that its automation would have on the entire systematic review process, summarize the existing information system support for each task, and highlight where further research is needed for realizing automation for the task. Integration of the systems that automate systematic review tasks may lead to a revised systematic review workflow. We envisage the optimized workflow will lead to system in which each systematic review is described as a computer program that automatically retrieves relevant trials, appraises them, extracts and synthesizes data, evaluates the risk of bias, performs meta-analysis calculations, and produces a report in real time. PMID:25005128

  11. Intermittency as a universal characteristic of the complete chromosome DNA sequences of eukaryotes: From protozoa to human genomes

    Science.gov (United States)

    Rybalko, S.; Larionov, S.; Poptsova, M.; Loskutov, A.

    2011-10-01

    Large-scale dynamical properties of complete chromosome DNA sequences of eukaryotes are considered. Using the proposed deterministic models with intermittency and symbolic dynamics we describe a wide spectrum of large-scale patterns inherent in these sequences, such as segmental duplications, tandem repeats, and other complex sequence structures. It is shown that the recently discovered gene number balance on the strands is not of a random nature, and certain subsystems of a complete chromosome DNA sequence exhibit the properties of deterministic chaos.

  12. Unicellular eukaryotes as models in cell and molecular biology: critical appraisal of their past and future value.

    Science.gov (United States)

    Simon, Martin; Plattner, Helmut

    2014-01-01

    Unicellular eukaryotes have been appreciated as model systems for the analysis of crucial questions in cell and molecular biology. This includes Dictyostelium (chemotaxis, amoeboid movement, phagocytosis), Tetrahymena (telomere structure, telomerase function), Paramecium (variant surface antigens, exocytosis, phagocytosis cycle) or both ciliates (ciliary beat regulation, surface pattern formation), Chlamydomonas (flagellar biogenesis and beat), and yeast (S. cerevisiae) for innumerable aspects. Nowadays many problems may be tackled with "higher" eukaryotic/metazoan cells for which full genomic information as well as domain databases, etc., were available long before protozoa. Established molecular tools, commercial antibodies, and established pharmacology are additional advantages available for higher eukaryotic cells. Moreover, an increasing number of inherited genetic disturbances in humans have become elucidated and can serve as new models. Among lower eukaryotes, yeast will remain a standard model because of its peculiarities, including its reduced genome and availability in the haploid form. But do protists still have a future as models? This touches not only the basic understanding of biology but also practical aspects of research, such as fund raising. As we try to scrutinize, due to specific advantages some protozoa should and will remain favorable models for analyzing novel genes or specific aspects of cell structure and function. Outstanding examples are epigenetic phenomena-a field of rising interest.

  13. Neutral Theory Predicts the Relative Abundance and Diversity of Genetic Elements in a Broad Array of Eukaryotic Genomes

    Science.gov (United States)

    Serra, François; Becher, Verónica; Dopazo, Hernán

    2013-01-01

    It is universally true in ecological communities, terrestrial or aquatic, temperate or tropical, that some species are very abundant, others are moderately common, and the majority are rare. Likewise, eukaryotic genomes also contain classes or “species” of genetic elements that vary greatly in abundance: DNA transposons, retrotransposons, satellite sequences, simple repeats and their less abundant functional sequences such as RNA or genes. Are the patterns of relative species abundance and diversity similar among ecological communities and genomes? Previous dynamical models of genomic diversity have focused on the selective forces shaping the abundance and diversity of transposable elements (TEs). However, ideally, models of genome dynamics should consider not only TEs, but also the diversity of all genetic classes or “species” populating eukaryotic genomes. Here, in an analysis of the diversity and abundance of genetic elements in >500 eukaryotic chromosomes, we show that the patterns are consistent with a neutral hypothesis of genome assembly in virtually all chromosomes tested. The distributions of relative abundance of genetic elements are quite precisely predicted by the dynamics of an ecological model for which the principle of functional equivalence is the main assumption. We hypothesize that at large temporal scales an overarching neutral or nearly neutral process governs the evolution of abundance and diversity of genetic elements in eukaryotic genomes. PMID:23798991

  14. Chef infrastructure automation cookbook

    CERN Document Server

    Marschall, Matthias

    2013-01-01

    Chef Infrastructure Automation Cookbook contains practical recipes on everything you will need to automate your infrastructure using Chef. The book is packed with illustrated code examples to automate your server and cloud infrastructure.The book first shows you the simplest way to achieve a certain task. Then it explains every step in detail, so that you can build your knowledge about how things work. Eventually, the book shows you additional things to consider for each approach. That way, you can learn step-by-step and build profound knowledge on how to go about your configuration management

  15. Alternative splicing: a pivotal step between eukaryotic transcription and translation.

    Science.gov (United States)

    Kornblihtt, Alberto R; Schor, Ignacio E; Alló, Mariano; Dujardin, Gwendal; Petrillo, Ezequiel; Muñoz, Manuel J

    2013-03-01

    Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

  16. Regulation of eukaryotic DNA replication and nuclear structure

    Institute of Scientific and Technical Information of China (English)

    WUJIARUI

    1999-01-01

    In eukaryote,nuclear structure is a key component for the functions of eukaryotic cells.More and more evidences show that the nuclear structure plays important role in regulating DNA replication.The nuclear structure provides a physical barrier for the replication licensing,participates in the decision where DNA replication initiates,and organizes replication proteins as replication factory for DNA replication.Through these works,new concepts on the regulation of DNA replication have emerged,which will be discussed in this minireview.

  17. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

    Science.gov (United States)

    Riechmann, J L; Heard, J; Martin, G; Reuber, L; Jiang, C; Keddie, J; Adam, L; Pineda, O; Ratcliffe, O J; Samaha, R R; Creelman, R; Pilgrim, M; Broun, P; Zhang, J Z; Ghandehari, D; Sherman, B K; Yu, G

    2000-12