diArk – a resource for eukaryotic genome research

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Kollmar Martin

2007-04-01

Full Text Available Abstract Background The number of completed eukaryotic genome sequences and cDNA projects has increased exponentially in the past few years although most of them have not been published yet. In addition, many microarray analyses yielded thousands of sequenced EST and cDNA clones. For the researcher interested in single gene analyses (from a phylogenetic, a structural biology or other perspective it is therefore important to have up-to-date knowledge about the various resources providing primary data. Description The database is built around 3 central tables: species, sequencing projects and publications. The species table contains commonly and alternatively used scientific names, common names and the complete taxonomic information. For projects the sequence type and links to species project web-sites and species homepages are stored. All publications are linked to projects. The web-interface provides comprehensive search modules with detailed options and three different views of the selected data. We have especially focused on developing an elaborate taxonomic tree search tool that allows the user to instantaneously identify e.g. the closest relative to the organism of interest. Conclusion We have developed a database, called diArk, to store, organize, and present the most relevant information about completed genome projects and EST/cDNA data from eukaryotes. Currently, diArk provides information about 415 eukaryotes, 823 sequencing projects, and 248 publications.

GFFview: A Web Server for Parsing and Visualizing Annotation Information of Eukaryotic Genome.

Science.gov (United States)

Deng, Feilong; Chen, Shi-Yi; Wu, Zhou-Lin; Hu, Yongsong; Jia, Xianbo; Lai, Song-Jia

2017-10-01

Owing to wide application of RNA sequencing (RNA-seq) technology, more and more eukaryotic genomes have been extensively annotated, such as the gene structure, alternative splicing, and noncoding loci. Annotation information of genome is prevalently stored as plain text in General Feature Format (GFF), which could be hundreds or thousands Mb in size. Therefore, it is a challenge for manipulating GFF file for biologists who have no bioinformatic skill. In this study, we provide a web server (GFFview) for parsing the annotation information of eukaryotic genome and then generating statistical description of six indices for visualization. GFFview is very useful for investigating quality and difference of the de novo assembled transcriptome in RNA-seq studies.

The eukaryotic genome is structurally and functionally more like a social insect colony than a book.

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Qiu, Guo-Hua; Yang, Xiaoyan; Zheng, Xintian; Huang, Cuiqin

2017-11-01

Traditionally, the genome has been described as the 'book of life'. However, the metaphor of a book may not reflect the dynamic nature of the structure and function of the genome. In the eukaryotic genome, the number of centrally located protein-coding sequences is relatively constant across species, but the amount of noncoding DNA increases considerably with the increase of organismal evolutional complexity. Therefore, it has been hypothesized that the abundant peripheral noncoding DNA protects the genome and the central protein-coding sequences in the eukaryotic genome. Upon comparison with the habitation, sociality and defense mechanisms of a social insect colony, it is found that the genome is similar to a social insect colony in various aspects. A social insect colony may thus be a better metaphor than a book to describe the spatial organization and physical functions of the genome. The potential implications of the metaphor are also discussed.

Strategien zur HLA-Typisierung mit PyrosequencingTM

OpenAIRE

Entz, Patricia

2006-01-01

Der Haupthistokompatibilitätskomplex ist durch seine biologische Funktion eine für die Diagnostik und Forschung äußerst wichtige Region im humanen Genom. Die Untersuchung von HLA-Genorten stellt ein wichtiges Instrument in der molekulargenetischen Praxis dar. Die Pyrosequencing-Technik ist gut geeignet, um kurze DNA-Abschnitte mit weitgehend bekannter Sequenz schnell und effizient zu untersuchen. Ziel dieser Arbeit war die Entwicklung von Pyrosequencing-basierten Methoden zur HLA-Typisierung....

The COG database: an updated version includes eukaryotes

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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

An SVD-based comparison of nine whole eukaryotic genomes supports a coelomate rather than ecdysozoan lineage

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Stuart Gary W

2004-12-01

Full Text Available Abstract Background Eukaryotic whole genome sequences are accumulating at an impressive rate. Effective methods for comparing multiple whole eukaryotic genomes on a large scale are needed. Most attempted solutions involve the production of large scale alignments, and many of these require a high stringency pre-screen for putative orthologs in order to reduce the effective size of the dataset and provide a reasonably high but unknown fraction of correctly aligned homologous sites for comparison. As an alternative, highly efficient methods that do not require the pre-alignment of operationally defined orthologs are also being explored. Results A non-alignment method based on the Singular Value Decomposition (SVD was used to compare the predicted protein complement of nine whole eukaryotic genomes ranging from yeast to man. This analysis resulted in the simultaneous identification and definition of a large number of well conserved motifs and gene families, and produced a species tree supporting one of two conflicting hypotheses of metazoan relationships. Conclusions Our SVD-based analysis of the entire protein complement of nine whole eukaryotic genomes suggests that highly conserved motifs and gene families can be identified and effectively compared in a single coherent definition space for the easy extraction of gene and species trees. While this occurs without the explicit definition of orthologs or homologous sites, the analysis can provide a basis for these definitions.

Meeting Report: Minutes from EMBO: Ten Years of Comparative Genomics of Eukaryotic Microorganisms

Czech Academy of Sciences Publication Activity Database

Lukeš, Julius; López-García, P.; Louis, E.; Boekhout, T.

2016-01-01

Roč. 167, č. 3 (2016), s. 217-221 ISSN 1434-4610 Institutional support: RVO:60077344 Keywords : protist * eukaryotic microorganisms * genomics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.794, year: 2016

Visualization of genome signatures of eukaryote genomes by batch-learning self-organizing map with a special emphasis on Drosophila genomes.

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Abe, Takashi; Hamano, Yuta; Ikemura, Toshimichi

2014-01-01

A strategy of evolutionary studies that can compare vast numbers of genome sequences is becoming increasingly important with the remarkable progress of high-throughput DNA sequencing methods. We previously established a sequence alignment-free clustering method "BLSOM" for di-, tri-, and tetranucleotide compositions in genome sequences, which can characterize sequence characteristics (genome signatures) of a wide range of species. In the present study, we generated BLSOMs for tetra- and pentanucleotide compositions in approximately one million sequence fragments derived from 101 eukaryotes, for which almost complete genome sequences were available. BLSOM recognized phylotype-specific characteristics (e.g., key combinations of oligonucleotide frequencies) in the genome sequences, permitting phylotype-specific clustering of the sequences without any information regarding the species. In our detailed examination of 12 Drosophila species, the correlation between their phylogenetic classification and the classification on the BLSOMs was observed to visualize oligonucleotides diagnostic for species-specific clustering.

Characteristics of 454 pyrosequencing data--enabling realistic simulation with flowsim.

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Balzer, Susanne; Malde, Ketil; Lanzén, Anders; Sharma, Animesh; Jonassen, Inge

2010-09-15

The commercial launch of 454 pyrosequencing in 2005 was a milestone in genome sequencing in terms of performance and cost. Throughout the three available releases, average read lengths have increased to approximately 500 base pairs and are thus approaching read lengths obtained from traditional Sanger sequencing. Study design of sequencing projects would benefit from being able to simulate experiments. We explore 454 raw data to investigate its characteristics and derive empirical distributions for the flow values generated by pyrosequencing. Based on our findings, we implement Flowsim, a simulator that generates realistic pyrosequencing data files of arbitrary size from a given set of input DNA sequences. We finally use our simulator to examine the impact of sequence lengths on the results of concrete whole-genome assemblies, and we suggest its use in planning of sequencing projects, benchmarking of assembly methods and other fields. Flowsim is freely available under the General Public License from http://blog.malde.org/index.php/flowsim/.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

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Brosius, Jürgen

2014-01-01

Currently, the best scenario for earliest forms of life is based on RNA molecules as they have the proven ability to catalyze enzymatic reactions and harbor genetic information. Evolutionary principles valid today become apparent in such models already. Furthermore, many features of eukaryotic genome architecture might have their origins in an RNA or RNA/protein (RNP) world, including the onset of a further transition, when DNA replaced RNA as the genetic bookkeeper of the cell. Chromosome maintenance, splicing, and regulatory function via RNA may be deeply rooted in the RNA/RNP worlds. Mostly in eukaryotes, conversion from RNA to DNA is still ongoing, which greatly impacts the plasticity of extant genomes. Raw material for novel genes encoding protein or RNA, or parts of genes including regulatory elements that selection can act on, continues to enter the evolutionary lottery. PMID:25081515

Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

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Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

2013-01-01

BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

Comparisons of the fungal and protistan communities among different marine sponge holobionts by pyrosequencing.

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He, Liming; Liu, Fang; Karuppiah, Valliappan; Ren, Yi; Li, Zhiyong

2014-05-01

To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.

EuGI: a novel resource for studying genomic islands to facilitate horizontal gene transfer detection in eukaryotes.

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Clasen, Frederick Johannes; Pierneef, Rian Ewald; Slippers, Bernard; Reva, Oleg

2018-05-03

Genomic islands (GIs) are inserts of foreign DNA that have potentially arisen through horizontal gene transfer (HGT). There are evidences that GIs can contribute significantly to the evolution of prokaryotes. The acquisition of GIs through HGT in eukaryotes has, however, been largely unexplored. In this study, the previously developed GI prediction tool, SeqWord Gene Island Sniffer (SWGIS), is modified to predict GIs in eukaryotic chromosomes. Artificial simulations are used to estimate ratios of predicting false positive and false negative GIs by inserting GIs into different test chromosomes and performing the SWGIS v2.0 algorithm. Using SWGIS v2.0, GIs are then identified in 36 fungal, 22 protozoan and 8 invertebrate genomes. SWGIS v2.0 predicts GIs in large eukaryotic chromosomes based on the atypical nucleotide composition of these regions. Averages for predicting false negative and false positive GIs were 20.1% and 11.01% respectively. A total of 10,550 GIs were identified in 66 eukaryotic species with 5299 of these GIs coding for at least one functional protein. The EuGI web-resource, freely accessible at http://eugi.bi.up.ac.za , was developed that allows browsing the database created from identified GIs and genes within GIs through an interactive and visual interface. SWGIS v2.0 along with the EuGI database, which houses GIs identified in 66 different eukaryotic species, and the EuGI web-resource, provide the first comprehensive resource for studying HGT in eukaryotes.

The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation

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Blanc, Guillaume; Agarkova, Irina; Grimwood, Jane; Kuo, Alan; Brueggeman, Andrew; Dunigan, David D.; Gurnon, James; Ladunga, Istvan; Lindquist, Erika; Lucas, Susan; Pangilinan, Jasmyn; Proschold, Thomas; Salamov, Asaf; Schmutz, Jeremy; Weeks, Donald; Tamada, Takashi; Lomsadze, Alexandre; Borodovsky, Mark; Claverie, Jean-Michel; Grigoriev, Igor V.; Van Etten, James L.

2012-02-13

Background Little is known about the mechanisms of adaptation of life to the extreme environmental conditions encountered in polar regions. Here we present the genome sequence of a unicellular green alga from the division chlorophyta, Coccomyxa subellipsoidea C-169, which we will hereafter refer to as C-169. This is the first eukaryotic microorganism from a polar environment to have its genome sequenced. Results The 48.8 Mb genome contained in 20 chromosomes exhibits significant synteny conservation with the chromosomes of its relatives Chlorella variabilis and Chlamydomonas reinhardtii. The order of the genes is highly reshuffled within synteny blocks, suggesting that intra-chromosomal rearrangements were more prevalent than inter-chromosomal rearrangements. Remarkably, Zepp retrotransposons occur in clusters of nested elements with strictly one cluster per chromosome probably residing at the centromere. Several protein families overrepresented in C. subellipsoidae include proteins involved in lipid metabolism, transporters, cellulose synthases and short alcohol dehydrogenases. Conversely, C-169 lacks proteins that exist in all other sequenced chlorophytes, including components of the glycosyl phosphatidyl inositol anchoring system, pyruvate phosphate dikinase and the photosystem 1 reaction center subunit N (PsaN). Conclusions We suggest that some of these gene losses and gains could have contributed to adaptation to low temperatures. Comparison of these genomic features with the adaptive strategies of psychrophilic microbes suggests that prokaryotes and eukaryotes followed comparable evolutionary routes to adapt to cold environments.

Complete genome sequence of a novel Plum pox virus strain W isolate determined by 454 pyrosequencing.

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Sheveleva, Anna; Kudryavtseva, Anna; Speranskaya, Anna; Belenikin, Maxim; Melnikova, Natalia; Chirkov, Sergei

2013-10-01

The near-complete (99.7 %) genome sequence of a novel Russian Plum pox virus (PPV) isolate Pk, belonging to the strain Winona (W), has been determined by 454 pyrosequencing with the exception of the thirty-one 5'-terminal nucleotides. This region was amplified using 5'RACE kit and sequenced by the Sanger method. Genomic RNA released from immunocaptured PPV particles was employed for generation of cDNA library using TransPlex Whole transcriptome amplification kit (WTA2, Sigma-Aldrich). The entire Pk genome has identity level of 92.8-94.5 % when compared to the complete nucleotide sequences of other PPV-W isolates (W3174, LV-141pl, LV-145bt, and UKR 44189), confirming a high degree of variability within the PPV-W strain. The isolates Pk and LV-141pl are most closely related. The Pk has been found in a wild plum (Prunus domestica) in a new region of Russia indicating widespread dissemination of the PPV-W strain in the European part of the former USSR.

Leucine-Rich repeat receptor kinases are sporadically distributed in eukaryotic genomes

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Diévart Anne

2011-12-01

Full Text Available Abstract Background Plant leucine-rich repeat receptor-like kinases (LRR-RLKs are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs. Results We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle. Conclusions In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.

Discrepancy variation of dinucleotide microsatellite repeats in eukaryotic genomes

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HUAN GAO

2009-01-01

Full Text Available To address whether there are differences of variation among repeat motif types and among taxonomic groups, we present here an analysis of variation and correlation of dinucleotide microsatellite repeats in eukaryotic genomes. Ten taxonomic groups were compared, those being primates, mammalia (excluding primates and rodentia, rodentia, birds, fish, amphibians and reptiles, insects, molluscs, plants and fungi, respectively. The data used in the analysis is from the literature published in the Journal of Molecular Ecology Notes. Analysis of variation reveals that there are no significant differences between AC and AG repeat motif types. Moreover, the number of alleles correlates positively with the copy number in both AG and AC repeats. Similar conclusions can be obtained from each taxonomic group. These results strongly suggest that the increase of SSR variation is almost linear with the increase of the copy number of each repeat motif. As well, the results suggest that the variability of SSR in the genomes of low-ranking species seem to be more than that of high-ranking species, excluding primates and fungi.

C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families

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Cutler Sean R

2007-06-01

Full Text Available Abstract Background The carboxy termini of proteins are a frequent site of activity for a variety of biologically important functions, ranging from post-translational modification to protein targeting. Several short peptide motifs involved in protein sorting roles and dependent upon their proximity to the C-terminus for proper function have already been characterized. As a limited number of such motifs have been identified, the potential exists for genome-wide statistical analysis and comparative genomics to reveal novel peptide signatures functioning in a C-terminal dependent manner. We have applied a novel methodology to the prediction of C-terminal-anchored peptide motifs involving a simple z-statistic and several techniques for improving the signal-to-noise ratio. Results We examined the statistical over-representation of position-specific C-terminal tripeptides in 7 eukaryotic proteomes. Sequence randomization models and simple-sequence masking were applied to the successful reduction of background noise. Similarly, as C-terminal homology among members of large protein families may artificially inflate tripeptide counts in an irrelevant and obfuscating manner, gene-family clustering was performed prior to the analysis in order to assess tripeptide over-representation across protein families as opposed to across all proteins. Finally, comparative genomics was used to identify tripeptides significantly occurring in multiple species. This approach has been able to predict, to our knowledge, all C-terminally anchored targeting motifs present in the literature. These include the PTS1 peroxisomal targeting signal (SKL*, the ER-retention signal (K/HDEL*, the ER-retrieval signal for membrane bound proteins (KKxx*, the prenylation signal (CC* and the CaaX box prenylation motif. In addition to a high statistical over-representation of these known motifs, a collection of significant tripeptides with a high propensity for biological function exists

Neural Network Prediction of Translation Initiation Sites in Eukaryotes: Perspectives for EST and Genome analysis

DEFF Research Database (Denmark)

Pedersen, Anders Gorm; Nielsen, Henrik

1997-01-01

Translation in eukaryotes does not always start at the first AUG in an mRNA, implying that context information also plays a role.This makes prediction of translation initiation sites a non-trivial task, especially when analysing EST and genome data where the entire mature mRNA sequence is not known...

An HMM-based comparative genomic framework for detecting introgression in eukaryotes.

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Kevin J Liu

2014-06-01

Full Text Available One outcome of interspecific hybridization and subsequent effects of evolutionary forces is introgression, which is the integration of genetic material from one species into the genome of an individual in another species. The evolution of several groups of eukaryotic species has involved hybridization, and cases of adaptation through introgression have been already established. In this work, we report on PhyloNet-HMM-a new comparative genomic framework for detecting introgression in genomes. PhyloNet-HMM combines phylogenetic networks with hidden Markov models (HMMs to simultaneously capture the (potentially reticulate evolutionary history of the genomes and dependencies within genomes. A novel aspect of our work is that it also accounts for incomplete lineage sorting and dependence across loci. Application of our model to variation data from chromosome 7 in the mouse (Mus musculus domesticus genome detected a recently reported adaptive introgression event involving the rodent poison resistance gene Vkorc1, in addition to other newly detected introgressed genomic regions. Based on our analysis, it is estimated that about 9% of all sites within chromosome 7 are of introgressive origin (these cover about 13 Mbp of chromosome 7, and over 300 genes. Further, our model detected no introgression in a negative control data set. We also found that our model accurately detected introgression and other evolutionary processes from synthetic data sets simulated under the coalescent model with recombination, isolation, and migration. Our work provides a powerful framework for systematic analysis of introgression while simultaneously accounting for dependence across sites, point mutations, recombination, and ancestral polymorphism.

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development.

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Shima, Naoko; Pederson, Kayla D

2017-08-01

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploring repetitive DNA landscapes using REPCLASS, a tool that automates the classification of transposable elements in eukaryotic genomes.

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Feschotte, Cédric; Keswani, Umeshkumar; Ranganathan, Nirmal; Guibotsy, Marcel L; Levine, David

2009-07-23

Eukaryotic genomes contain large amount of repetitive DNA, most of which is derived from transposable elements (TEs). Progress has been made to develop computational tools for ab initio identification of repeat families, but there is an urgent need to develop tools to automate the annotation of TEs in genome sequences. Here we introduce REPCLASS, a tool that automates the classification of TE sequences. Using control repeat libraries, we show that the program can classify accurately virtually any known TE types. Combining REPCLASS to ab initio repeat finding in the genomes of Caenorhabditis elegans and Drosophila melanogaster allowed us to recover the contrasting TE landscape characteristic of these species. Unexpectedly, REPCLASS also uncovered several novel TE families in both genomes, augmenting the TE repertoire of these model species. When applied to the genomes of distant Caenorhabditis and Drosophila species, the approach revealed a remarkable conservation of TE composition profile within each genus, despite substantial interspecific covariations in genome size and in the number of TEs and TE families. Lastly, we applied REPCLASS to analyze 10 fungal genomes from a wide taxonomic range, most of which have not been analyzed for TE content previously. The results showed that TE diversity varies widely across the fungi "kingdom" and appears to positively correlate with genome size, in particular for DNA transposons. Together, these data validate REPCLASS as a powerful tool to explore the repetitive DNA landscapes of eukaryotes and to shed light onto the evolutionary forces shaping TE diversity and genome architecture.

Yeast 2.0-connecting the dots in the construction of the world's first functional synthetic eukaryotic genome.

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Pretorius, I S; Boeke, J D

2018-06-01

Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the

Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs with an emphasis on poplar

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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

Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes.

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Lacroix, Benoît; Citovsky, Vitaly

2018-03-03

Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host-pathogen interactions

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Laura eGomez-Valero

2011-10-01

Full Text Available Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic-like proteins, many of which have been shown to modulate host cell functions to the pathogen's advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed.

Transfer of DNA from Bacteria to Eukaryotes

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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.

DNA to DNA transcription might exist in eukaryotic cells

OpenAIRE

Li, Gao-De

2016-01-01

Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes

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Marsh, T. L.; Reich, C. I.; Whitelock, R. B.; Olsen, G. J.; Woese, C. R. (Principal Investigator)

1994-01-01

The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.

Long-Range Order and Fractality in the Structure and Organization of Eukaryotic Genomes

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Polychronopoulos, Dimitris; Tsiagkas, Giannis; Athanasopoulou, Labrini; Sellis, Diamantis; Almirantis, Yannis

2014-12-01

The late Professor J.S. Nicolis always emphasized, both in his writings and in presentations and discussions with students and friends, the relevance of a dynamical systems approach to biology. In particular, viewing the genome as a "biological text" captures the dynamical character of both the evolution and function of the organisms in the form of correlations indicating the presence of a long-range order. This genomic structure can be expressed in forms reminiscent of natural languages and several temporal and spatial traces l by the functioning of dynamical systems: Zipf laws, self-similarity and fractality. Here we review several works of our group and recent unpublished results, focusing on the chromosomal distribution of biologically active genomic components: Genes and protein-coding segments, CpG islands, transposable elements belonging to all major classes and several types of conserved non-coding genomic elements. We report the systematic appearance of power-laws in the size distribution of the distances between elements belonging to each of these types of functional genomic elements. Moreover, fractality is also found in several cases, using box-counting and entropic scaling.We present here, for the first time in a unified way, an aggregative model of the genomic dynamics which can explain the observed patterns on the grounds of known phenomena accompanying genome evolution. Our results comply with recent findings about a "fractal globule" geometry of chromatin in the eukaryotic nucleus.

Pyrosequencing reveals diverse microbial community associated with the zoanthid Palythoa australiae from the South China Sea.

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Sun, Wei; Zhang, Fengli; He, Liming; Li, Zhiyong

2014-05-01

Diverse sessile organisms inhabit the coral reef ecosystems, including corals, sponges, and sea anemones. In the past decades, scleractinian corals (Cnidaria, Anthozoa, Scleractinia) and their associated microorganisms have attracted much attention. Zoanthids (Cnidaria, Anthozoa, Zoanthidea) are commonly found in coral reefs. However, little is known about the community structure of zoanthid-associated microbiota. In this study, the microbial community associated with the zoanthid Palythoa australiae in the South China Sea was investigated by 454 pyrosequencing. As a result, 2,353 bacterial, 583 archaeal, and 36 eukaryotic microbial ribotypes were detected, respectively. A total of 22 bacterial phyla (16 formally described phyla and six candidate phyla) were recovered. Proteobacteria was the most abundant group, followed by Chloroflexi and Actinobacteria. High-abundance Rhizobiales and diverse Chloroflexi were observed in the bacterial community. The archaeal population was composed of Crenarchaeota and Euryarchaeota, with Marine Group I as the dominant lineage. In particular, Candidatus Nitrosopumilus dominated the archaeal community. Besides bacteria and archaea, the zoanthid harbored eukaryotic microorganisms including fungi and algae though their diversity was very low. This study provided the first insights into the microbial community associated with P. australiae by 454 pyrosequencing, consequently laid a basis for the understanding of the association of P. australiae-microbes symbioses.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

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Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes.

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Moriyama, Takashi; Sato, Naoki

2014-01-01

Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.

Deep sequencing of subseafloor eukaryotic rRNA reveals active Fungi across marine subsurface provinces.

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William Orsi

Full Text Available The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC, nitrate, sulfide, and dissolved inorganic carbon (DIC. These correlations are supported by terminal restriction length polymorphism (TRFLP analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

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Bonten Marc JM

2010-04-01

Full Text Available Abstract Background The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. Results We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI, which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. Conclusions Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.

How natural a kind is "eukaryote?".

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Doolittle, W Ford

2014-06-02

Systematics balances uneasily between realism and nominalism, uncommitted as to whether biological taxa are discoveries or inventions. If the former, they might be taken as natural kinds. I briefly review some philosophers' concepts of natural kinds and then argue that several of these apply well enough to "eukaryote." Although there are some sticky issues around genomic chimerism and when eukaryotes first appeared, if we allow for degrees in the naturalness of kinds, existing eukaryotes rank highly, higher than prokaryotes. Most biologists feel this intuitively: All I attempt to do here is provide some conceptual justification. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

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.

Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia.

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Zhang, Yan-Qiong; Chen, Dong-Liang; Tian, Hai-Feng; Zhang, Bao-Hong; Wen, Jian-Fan

2009-10-01

Using a combined computational program, we identified 50 potential microRNAs (miRNAs) in Giardia lamblia, one of the most primitive unicellular eukaryotes. These miRNAs are unique to G. lamblia and no homologues have been found in other organisms; miRNAs, currently known in other species, were not found in G. lamblia. This suggests that miRNA biogenesis and miRNA-mediated gene regulation pathway may evolve independently, especially in evolutionarily distant lineages. A majority (43) of the predicted miRNAs are located at one single locus; however, some miRNAs have two or more copies in the genome. Among the 58 miRNA genes, 28 are located in the intergenic regions whereas 30 are present in the anti-sense strands of the protein-coding sequences. Five predicted miRNAs are expressed in G. lamblia trophozoite cells evidenced by expressed sequence tags or RT-PCR. Thirty-seven identified miRNAs may target 50 protein-coding genes, including seven variant-specific surface proteins (VSPs). Our findings provide a clue that miRNA-mediated gene regulation may exist in the early stage of eukaryotic evolution, suggesting that it is an important regulation system ubiquitous in eukaryotes.

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

DEFF Research Database (Denmark)

Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris

2016-01-01

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

DEFF Research Database (Denmark)

Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris

2016-01-01

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for d...

Maximum likelihood phylogenetic reconstruction from high-resolution whole-genome data and a tree of 68 eukaryotes.

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Lin, Yu; Hu, Fei; Tang, Jijun; Moret, Bernard M E

2013-01-01

The rapid accumulation of whole-genome data has renewed interest in the study of the evolution of genomic architecture, under such events as rearrangements, duplications, losses. Comparative genomics, evolutionary biology, and cancer research all require tools to elucidate the mechanisms, history, and consequences of those evolutionary events, while phylogenetics could use whole-genome data to enhance its picture of the Tree of Life. Current approaches in the area of phylogenetic analysis are limited to very small collections of closely related genomes using low-resolution data (typically a few hundred syntenic blocks); moreover, these approaches typically do not include duplication and loss events. We describe a maximum likelihood (ML) approach for phylogenetic analysis that takes into account genome rearrangements as well as duplications, insertions, and losses. Our approach can handle high-resolution genomes (with 40,000 or more markers) and can use in the same analysis genomes with very different numbers of markers. Because our approach uses a standard ML reconstruction program (RAxML), it scales up to large trees. We present the results of extensive testing on both simulated and real data showing that our approach returns very accurate results very quickly. In particular, we analyze a dataset of 68 high-resolution eukaryotic genomes, with from 3,000 to 42,000 genes, from the eGOB database; the analysis, including bootstrapping, takes just 3 hours on a desktop system and returns a tree in agreement with all well supported branches, while also suggesting resolutions for some disputed placements.

The candidate phylum Poribacteria by single-cell genomics: new insights into phylogeny, cell-compartmentation, eukaryote-like repeat proteins, and other genomic features.

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Janine Kamke

Full Text Available The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake.

Positive selection for unpreferred codon usage in eukaryotic genomes

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Galagan James E

2007-07-01

Full Text Available Abstract Background Natural selection has traditionally been understood as a force responsible for pushing genes to states of higher translational efficiency, whereas lower translational efficiency has been explained by neutral mutation and genetic drift. We looked for evidence of directional selection resulting in increased unpreferred codon usage (and presumably reduced translational efficiency in three divergent clusters of eukaryotic genomes using a simple optimal-codon-based metric (Kp/Ku. Results Here we show that for some genes natural selection is indeed responsible for causing accelerated unpreferred codon substitution, and document the scope of this selection. In Cryptococcus and to a lesser extent Drosophila, we find many genes showing a statistically significant signal of selection for unpreferred codon usage in one or more lineages. We did not find evidence for this type of selection in Saccharomyces. The signal of positive selection observed from unpreferred synonymous codon substitutions is coincident in Cryptococcus and Drosophila with the distribution of upstream open reading frames (uORFs, another genic feature known to reduce translational efficiency. Functional enrichment analysis of genes exhibiting low Kp/Ku ratios reveals that genes in regulatory roles are particularly subject to this type of selection. Conclusion Through genome-wide scans, we find recent selection for unpreferred codon usage at approximately 1% of genetic loci in a Cryptococcus and several genes in Drosophila. Unpreferred codons can impede translation efficiency, and we find that genes with translation-impeding uORFs are enriched for this selection signal. We find that regulatory genes are particularly likely to be subject to selection for unpreferred codon usage. Given that expression noise can propagate through regulatory cascades, and that low translational efficiency can reduce expression noise, this finding supports the hypothesis that translational

Intermediary metabolism in protists: a sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes.

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Ginger, Michael L; Fritz-Laylin, Lillian K; Fulton, Chandler; Cande, W Zacheus; Dawson, Scott C

2010-12-01

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. Copyright © 2010 Elsevier GmbH. All rights reserved.

Eukaryotic acquisition of a bacterial operon

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The yeast Saccharomyces cerevisiae is one of the champions of basic biomedical research due to its compact eukaryotic genome and ease of experimental manipulation. Despite these immense strengths, its impact on understanding the genetic basis of natural phenotypic variation has been limited by strai...

Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

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Atteia, Ariane; van Lis, Robert; Tielens, Aloysius G M; Martin, William F

2013-02-01

Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems. Copyright © 2012 Elsevier B.V. All rights reserved.

Eukaryotic systematics: a user's guide for cell biologists and parasitologists.

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Walker, Giselle; Dorrell, Richard G; Schlacht, Alexander; Dacks, Joel B

2011-11-01

Single-celled parasites like Entamoeba, Trypanosoma, Phytophthora and Plasmodium wreak untold havoc on human habitat and health. Understanding the position of the various protistan pathogens in the larger context of eukaryotic diversity informs our study of how these parasites operate on a cellular level, as well as how they have evolved. Here, we review the literature that has brought our understanding of eukaryotic relationships from an idea of parasites as primitive cells to a crystallized view of diversity that encompasses 6 major divisions, or supergroups, of eukaryotes. We provide an updated taxonomic scheme (for 2011), based on extensive genomic, ultrastructural and phylogenetic evidence, with three differing levels of taxonomic detail for ease of referencing and accessibility (see supplementary material at Cambridge Journals On-line). Two of the most pressing issues in cellular evolution, the root of the eukaryotic tree and the evolution of photosynthesis in complex algae, are also discussed along with ideas about what the new generation of genome sequencing technologies may contribute to the field of eukaryotic systematics. We hope that, armed with this user's guide, cell biologists and parasitologists will be encouraged about taking an increasingly evolutionary point of view in the battle against parasites representing real dangers to our livelihoods and lives.

The diversity and structure of marine protists in the coastal waters of China revealed by morphological observation and 454 pyrosequencing

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Liu, Yun; Song, Shuqun; Chen, Tiantian; Li, Caiwen

2017-04-01

Pyrosequencing of the 18S rRNA gene has been widely adopted to study the eukaryotic diversity in various types of environments, and has an advantage over traditional morphology methods in exploring unknown microbial communities. To comprehensively assess the diversity and community composition of marine protists in the coastal waters of China, we applied both morphological observations and high-throughput sequencing of the V2 and V3 regions of 18S rDNA simultaneously to analyze samples collected from the surface layer of the Yellow and East China Seas. Dinoflagellates, diatoms and ciliates were the three dominant protistan groups as revealed by the two methods. Diatoms were the first dominant protistan group in the microscopic observations, with Skeletonema mainly distributed in the nearshore eutrophic waters and Chaetoceros in higher temperature and higher pH waters. The mixotrophic dinoflagellates, Gymnodinium and Gyrodinium, were more competitive in the oligotrophic waters. The pyrosequencing method revealed an extensive diversity of dinoflagellates. Chaetoceros was the only dominant diatom group in the pyrosequencing dataset. Gyrodinium represented the most abundant reads and dominated the offshore oligotrophic protistan community as they were in the microscopic observations. The dominance of parasitic dinoflagellates in the pyrosequencing dataset, which were overlooked in the morphological observations, indicates more attention should be paid to explore the potential role of this group. Both methods provide coherent clustering of samples. Nutrient levels, salinity and pH were the main factors influencing the distribution of protists. This study demonstrates that different primer pairs used in the pyrosequencing will indicate different protistan community structures. A suitable marker may reveal more comprehensive composition of protists and provide valuable information on environmental drivers.

Large variability of bathypelagic microbial eukaryotic communities across the world's oceans.

Science.gov (United States)

Pernice, Massimo C; Giner, Caterina R; Logares, Ramiro; Perera-Bel, Júlia; Acinas, Silvia G; Duarte, Carlos M; Gasol, Josep M; Massana, Ramon

2016-04-01

In this work, we study the diversity of bathypelagic microbial eukaryotes (0.8-20 μm) in the global ocean. Seawater samples from 3000 to 4000 m depth from 27 stations in the Atlantic, Pacific and Indian Oceans were analyzed by pyrosequencing the V4 region of the 18S ribosomal DNA. The relative abundance of the most abundant operational taxonomic units agreed with the results of a parallel metagenomic analysis, suggesting limited PCR biases in the tag approach. Although rarefaction curves for single stations were seldom saturated, the global analysis of all sequences together suggested an adequate recovery of bathypelagic diversity. Community composition presented a large variability among samples, which was poorly explained by linear geographic distance. In fact, the similarity between communities was better explained by water mass composition (26% of the variability) and the ratio in cell abundance between prokaryotes and microbial eukaryotes (21%). Deep diversity appeared dominated by four taxonomic groups (Collodaria, Chrysophytes, Basidiomycota and MALV-II) appearing in different proportions in each sample. Novel diversity amounted to 1% of the pyrotags and was lower than expected. Our study represents an essential step in the investigation of bathypelagic microbial eukaryotes, indicating dominating taxonomic groups and suggesting idiosyncratic assemblages in distinct oceanic regions.

Consistent mutational paths predict eukaryotic thermostability

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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.

Origins and evolution of viruses of eukaryotes: The ultimate modularity

International Nuclear Information System (INIS)

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

2015-01-01

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

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

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.

INVESTIGATIONS INTO MOLECULAR PATHWAYS IN THE POST GENOME ERA: CROSS SPECIES COMPARATIVE GENOMICS APPROACH

Science.gov (United States)

Genome sequencing efforts in the past decade were aimed at generating draft sequences of many prokaryotic and eukaryotic model organisms. Successful completion of unicellular eukaryotes, worm, fly and human genome have opened up the new field of molecular biology and function...

Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river.

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Bricheux, Geneviève; Morin, Loïc; Le Moal, Gwenaël; Coffe, Gérard; Balestrino, Damien; Charbonnel, Nicolas; Bohatier, Jacques; Forestier, Christiane

2013-06-01

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Lateral gene transfer between prokaryotes and multicellular eukaryotes: ongoing and significant?

NARCIS (Netherlands)

Ros, V.I.D.; Hurst, G.D.D.

2009-01-01

The expansion of genome sequencing projects has produced accumulating evidence for lateral transfer of genes between prokaryotic and eukaryotic genomes. However, it remains controversial whether these genes are of functional importance in their recipient host. Nikoh and Nakabachi, in a recent paper

Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

Science.gov (United States)

Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H

2010-02-01

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

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How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

2015-07-01

Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

The Eukaryotic Pathogen Databases: a functional genomic resource integrating data from human and veterinary parasites.

Science.gov (United States)

Harb, Omar S; Roos, David S

2015-01-01

Over the past 20 years, advances in high-throughput biological techniques and the availability of computational resources including fast Internet access have resulted in an explosion of large genome-scale data sets "big data." While such data are readily available for download and personal use and analysis from a variety of repositories, often such analysis requires access to seldom-available computational skills. As a result a number of databases have emerged to provide scientists with online tools enabling the interrogation of data without the need for sophisticated computational skills beyond basic knowledge of Internet browser utility. This chapter focuses on the Eukaryotic Pathogen Databases (EuPathDB: http://eupathdb.org) Bioinformatic Resource Center (BRC) and illustrates some of the available tools and methods.

Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

NARCIS (Netherlands)

Yardimci, Hasan; Loveland, Anna B.; Habuchi, Satoshi; van Oijen, Antoine M.; Walter, Johannes C.

2010-01-01

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes.

454 pyrosequencing to describe microbial eukaryotic community composition, diversity and relative abundance: a test for marine haptophytes.

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Elianne Egge

Full Text Available Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000-20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.

Atypical mitochondrial inheritance patterns in eukaryotes.

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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.

Large variability of bathypelagic microbial eukaryotic communities across the world’s oceans

KAUST Repository

Pernice, Massimo C.

2015-10-09

In this work, we study the diversity of bathypelagic microbial eukaryotes (0.8–20 μm) in the global ocean. Seawater samples from 3000 to 4000 m depth from 27 stations in the Atlantic, Pacific and Indian Oceans were analyzed by pyrosequencing the V4 region of the 18S ribosomal DNA. The relative abundance of the most abundant operational taxonomic units agreed with the results of a parallel metagenomic analysis, suggesting limited PCR biases in the tag approach. Although rarefaction curves for single stations were seldom saturated, the global analysis of all sequences together suggested an adequate recovery of bathypelagic diversity. Community composition presented a large variability among samples, which was poorly explained by linear geographic distance. In fact, the similarity between communities was better explained by water mass composition (26% of the variability) and the ratio in cell abundance between prokaryotes and microbial eukaryotes (21%). Deep diversity appeared dominated by four taxonomic groups (Collodaria, Chrysophytes, Basidiomycota and MALV-II) appearing in different proportions in each sample. Novel diversity amounted to 1% of the pyrotags and was lower than expected. Our study represents an essential step in the investigation of bathypelagic microbial eukaryotes, indicating dominating taxonomic groups and suggesting idiosyncratic assemblages in distinct oceanic regions.

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists

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Matheus Sanitá Lima

2017-11-01

Full Text Available Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb, indicating that most of the organelle DNA—coding and noncoding—is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells.

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

prokaryotic genome. Based on a protein alignment we could group the P5 ATPases into two subfamilies, P5A and P5B that, based on the number of negative charges in conserved trans-membrane segment 4, are likely to have different ion specificities. P5A ATPases are present in all eukaryotic genomes sequenced so......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...

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

Science.gov (United States)

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Characterization of the Zoarces viviparus liver transcriptome using massively parallel pyrosequencing

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Asker Noomi

2009-07-01

Full Text Available Abstract Background The teleost Zoarces viviparus (eelpout lives along the coasts of Northern Europe and has long been an established model organism for marine ecology and environmental monitoring. The scarce information about this species genome has however restrained the use of efficient molecular-level assays, such as gene expression microarrays. Results In the present study we present the first comprehensive characterization of the Zoarces viviparus liver transcriptome. From 400,000 reads generated by massively parallel pyrosequencing, more than 50,000 pieces of putative transcripts were assembled, annotated and functionally classified. The data was estimated to cover roughly 40% of the total transcriptome and homologues for about half of the genes of Gasterosteus aculeatus (stickleback were identified. The sequence data was consequently used to design an oligonucleotide microarray for large-scale gene expression analysis. Conclusion Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates enough genomic information for adequate de novo assembly of a large number of genes in a higher vertebrate. The generated sequence data, including the validated microarray probes, are publicly available to promote genome-wide research in Zoarces viviparus.

Genome Improvement at JGI-HAGSC

Energy Technology Data Exchange (ETDEWEB)

Grimwood, Jane; Schmutz, Jeremy J.; Myers, Richard M.

2012-03-03

Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence. For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.

Microplanktonic community structure in a coastal system relative to a Phaeocystis bloom inferred from morphological and tag pyrosequencing methods.

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Sébastien Monchy

Full Text Available BACKGROUND: Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC, have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom. METHODOLOGY AND PRINCIPAL FINDINGS: The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae. CONCLUSIONS: Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

Science.gov (United States)

Jagus, Rosemary; Bachvaroff, Tsvetan R; Joshi, Bhavesh; Place, Allen R

2012-01-01

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

On the Diversification of the Translation Apparatus across Eukaryotes

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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.

Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

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Shcherbik, Svetlana V; Pearce, Nicholas C; Levine, Marnie L; Klimov, Alexander I; Villanueva, Julie M; Bousse, Tatiana L

2014-01-01

Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.

Directory of Open Access Journals (Sweden)

Svetlana V Shcherbik

Full Text Available BACKGROUND: Live attenuated influenza vaccine viruses (LAIVs can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV and a seasonal wild-type (wt virus. The vaccine candidates contain hemagglutinin (HA and neuraminidase (NA genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses. METHODOLOGY/PRINCIPAL FINDINGS: This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2 (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012 or influenza A (H7N9 (A/Anhui/1/2013 wt viruses with the MDV A/Leningrad/134/17/57(H2N2. Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

Efficient alignment of pyrosequencing reads for re-sequencing applications

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Russo Luis MS

2011-05-01

Full Text Available Abstract Background Over the past few years, new massively parallel DNA sequencing technologies have emerged. These platforms generate massive amounts of data per run, greatly reducing the cost of DNA sequencing. However, these techniques also raise important computational difficulties mostly due to the huge volume of data produced, but also because of some of their specific characteristics such as read length and sequencing errors. Among the most critical problems is that of efficiently and accurately mapping reads to a reference genome in the context of re-sequencing projects. Results We present an efficient method for the local alignment of pyrosequencing reads produced by the GS FLX (454 system against a reference sequence. Our approach explores the characteristics of the data in these re-sequencing applications and uses state of the art indexing techniques combined with a flexible seed-based approach, leading to a fast and accurate algorithm which needs very little user parameterization. An evaluation performed using real and simulated data shows that our proposed method outperforms a number of mainstream tools on the quantity and quality of successful alignments, as well as on the execution time. Conclusions The proposed methodology was implemented in a software tool called TAPyR--Tool for the Alignment of Pyrosequencing Reads--which is publicly available from http://www.tapyr.net.

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

Science.gov (United States)

Sanitá Lima, Matheus; Smith, David Roy

2017-11-06

Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb), indicating that most of the organelle DNA-coding and noncoding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017 Sanitá Lima and Smith.

An algorithm for detecting eukaryotic sequences in metagenomic ...

Indian Academy of Sciences (India)

species but also from accidental contamination from the genome of eukaryotic host cells. The latter scenario generally occurs in the case of host-associated metagenomes, e.g. microbes living in human gut. In such cases, one needs to identify and remove contaminating host DNA sequences, since the latter sequences will ...

In silico ionomics segregates parasitic from free-living eukaryotes.

Science.gov (United States)

Greganova, Eva; Steinmann, Michael; Mäser, Pascal; Fankhauser, Niklaus

2013-01-01

Ion transporters are fundamental to life. Due to their ancient origin and conservation in sequence, ion transporters are also particularly well suited for comparative genomics of distantly related species. Here, we perform genome-wide ion transporter profiling as a basis for comparative genomics of eukaryotes. From a given predicted proteome, we identify all bona fide ion channels, ion porters, and ion pumps. Concentrating on unicellular eukaryotes (n = 37), we demonstrate that clustering of species according to their repertoire of ion transporters segregates obligate endoparasites (n = 23) on the one hand, from free-living species and facultative parasites (n = 14) on the other hand. This surprising finding indicates strong convergent evolution of the parasites regarding the acquisition and homeostasis of inorganic ions. Random forest classification identifies transporters of ammonia, plus transporters of iron and other transition metals, as the most informative for distinguishing the obligate parasites. Thus, in silico ionomics further underscores the importance of iron in infection biology and suggests access to host sources of nitrogen and transition metals to be selective forces in the evolution of parasitism. This finding is in agreement with the phenomenon of iron withholding as a primordial antimicrobial strategy of infected mammals.

Origin and evolution of the self-organizing cytoskeleton in the network of eukaryotic organelles.

Science.gov (United States)

Jékely, Gáspár

2014-09-02

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing "active gel," the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

A Taste of Algal Genomes from the Joint Genome Institute

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Kuo, Alan; Grigoriev, Igor

2012-06-17

Algae play profound roles in aquatic food chains and the carbon cycle, can impose health and economic costs through toxic blooms, provide models for the study of symbiosis, photosynthesis, and eukaryotic evolution, and are candidate sources for bio-fuels; all of these research areas are part of the mission of DOE's Joint Genome Institute (JGI). To date JGI has sequenced, assembled, annotated, and released to the public the genomes of 18 species and strains of algae, sampling almost all of the major clades of photosynthetic eukaryotes. With more algal genomes currently undergoing analysis, JGI continues its commitment to driving forward basic and applied algal science. Among these ongoing projects are the pan-genome of the dominant coccolithophore Emiliania huxleyi, the interrelationships between the 4 genomes in the nucleomorph-containing Bigelowiella natans and Guillardia theta, and the search for symbiosis genes of lichens.

Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention.

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Johnston, Iain G; Williams, Ben P

2016-02-24

Since their endosymbiotic origin, mitochondria have lost most of their genes. Although many selective mechanisms underlying the evolution of mitochondrial genomes have been proposed, a data-driven exploration of these hypotheses is lacking, and a quantitatively supported consensus remains absent. We developed HyperTraPS, a methodology coupling stochastic modeling with Bayesian inference, to identify the ordering of evolutionary events and suggest their causes. Using 2015 complete mitochondrial genomes, we inferred evolutionary trajectories of mtDNA gene loss across the eukaryotic tree of life. We find that proteins comprising the structural cores of the electron transport chain are preferentially encoded within mitochondrial genomes across eukaryotes. A combination of high GC content and high protein hydrophobicity is required to explain patterns of mtDNA gene retention; a model that accounts for these selective pressures can also predict the success of artificial gene transfer experiments in vivo. This work provides a general method for data-driven inference of the ordering of evolutionary and progressive events, here identifying the distinct features shaping mitochondrial genomes of present-day species. Copyright © 2016 Elsevier Inc. All rights reserved.

On the Archaeal Origins of Eukaryotes and the Challenges of Inferring Phenotype from Genotype.

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Dey, Gautam; Thattai, Mukund; Baum, Buzz

2016-07-01

If eukaryotes arose through a merger between archaea and bacteria, what did the first true eukaryotic cell look like? A major step toward an answer came with the discovery of Lokiarchaeum, an archaeon whose genome encodes small GTPases related to those used by eukaryotes to regulate membrane traffic. Although 'Loki' cells have yet to be seen, their existence has prompted the suggestion that the archaeal ancestor of eukaryotes engulfed the future mitochondrion by phagocytosis. We propose instead that the archaeal ancestor was a relatively simple cell, and that eukaryotic cellular organization arose as the result of a gradual transfer of bacterial genes and membranes driven by an ever-closer symbiotic partnership between a bacterium and an archaeon. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Patterns of intron gain and conservation in eukaryotic genes

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Wolf Yuri I

2007-10-01

Full Text Available Abstract Background: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. Results: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. Conclusion: We obtained robust estimates of the contribution of parallel gain to the observed

Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance.

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Wen Xie

Full Text Available BACKGROUND: Bemisia tabaci (Gennadius is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. METHODOLOGY AND PRINCIPAL FINDINGS: Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45% unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10-5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. CONCLUSIONS: This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1.

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Elad Milrot

2017-08-01

Full Text Available A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1. Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.

RNase MRP and the RNA processing cascade in the eukaryotic ancestor.

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Woodhams, Michael D; Stadler, Peter F; Penny, David; Collins, Lesley J

2007-02-08

Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor. We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution. We conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

Archaeal “Dark Matter” and the Origin of Eukaryotes

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Williams, Tom A.; Embley, T. Martin

2014-01-01

Current hypotheses about the history of cellular life are mainly based on analyses of cultivated organisms, but these represent only a small fraction of extant biodiversity. The sequencing of new environmental lineages therefore provides an opportunity to test, revise, or reject existing ideas about the tree of life and the origin of eukaryotes. According to the textbook three domains hypothesis, the eukaryotes emerge as the sister group to a monophyletic Archaea. However, recent analyses incorporating better phylogenetic models and an improved sampling of the archaeal domain have generally supported the competing eocyte hypothesis, in which core genes of eukaryotic cells originated from within the Archaea, with important implications for eukaryogenesis. Given this trend, it was surprising that a recent analysis incorporating new genomes from uncultivated Archaea recovered a strongly supported three domains tree. Here, we show that this result was due in part to the use of a poorly fitting phylogenetic model and also to the inclusion by an automated pipeline of genes of putative bacterial origin rather than nucleocytosolic versions for some of the eukaryotes analyzed. When these issues were resolved, analyses including the new archaeal lineages placed core eukaryotic genes within the Archaea. These results are consistent with a number of recent studies in which improved archaeal sampling and better phylogenetic models agree in supporting the eocyte tree over the three domains hypothesis. PMID:24532674

pico-PLAZA, a genome database of microbial photosynthetic eukaryotes.

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Vandepoele, Klaas; Van Bel, Michiel; Richard, Guilhem; Van Landeghem, Sofie; Verhelst, Bram; Moreau, Hervé; Van de Peer, Yves; Grimsley, Nigel; Piganeau, Gwenael

2013-08-01

With the advent of next generation genome sequencing, the number of sequenced algal genomes and transcriptomes is rapidly growing. Although a few genome portals exist to browse individual genome sequences, exploring complete genome information from multiple species for the analysis of user-defined sequences or gene lists remains a major challenge. pico-PLAZA is a web-based resource (http://bioinformatics.psb.ugent.be/pico-plaza/) for algal genomics that combines different data types with intuitive tools to explore genomic diversity, perform integrative evolutionary sequence analysis and study gene functions. Apart from homologous gene families, multiple sequence alignments, phylogenetic trees, Gene Ontology, InterPro and text-mining functional annotations, different interactive viewers are available to study genome organization using gene collinearity and synteny information. Different search functions, documentation pages, export functions and an extensive glossary are available to guide non-expert scientists. To illustrate the versatility of the platform, different case studies are presented demonstrating how pico-PLAZA can be used to functionally characterize large-scale EST/RNA-Seq data sets and to perform environmental genomics. Functional enrichments analysis of 16 Phaeodactylum tricornutum transcriptome libraries offers a molecular view on diatom adaptation to different environments of ecological relevance. Furthermore, we show how complementary genomic data sources can easily be combined to identify marker genes to study the diversity and distribution of algal species, for example in metagenomes, or to quantify intraspecific diversity from environmental strains. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

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Roger Andrew J

2003-06-01

Full Text Available Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH, to evaluate and compare the patterns and rates of lateral gene transfer (LGT in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists. Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

Removing Noise From Pyrosequenced Amplicons

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Davenport Russell J

2011-01-01

Full Text Available Abstract Background In many environmental genomics applications a homologous region of DNA from a diverse sample is first amplified by PCR and then sequenced. The next generation sequencing technology, 454 pyrosequencing, has allowed much larger read numbers from PCR amplicons than ever before. This has revolutionised the study of microbial diversity as it is now possible to sequence a substantial fraction of the 16S rRNA genes in a community. However, there is a growing realisation that because of the large read numbers and the lack of consensus sequences it is vital to distinguish noise from true sequence diversity in this data. Otherwise this leads to inflated estimates of the number of types or operational taxonomic units (OTUs present. Three sources of error are important: sequencing error, PCR single base substitutions and PCR chimeras. We present AmpliconNoise, a development of the PyroNoise algorithm that is capable of separately removing 454 sequencing errors and PCR single base errors. We also introduce a novel chimera removal program, Perseus, that exploits the sequence abundances associated with pyrosequencing data. We use data sets where samples of known diversity have been amplified and sequenced to quantify the effect of each of the sources of error on OTU inflation and to validate these algorithms. Results AmpliconNoise outperforms alternative algorithms substantially reducing per base error rates for both the GS FLX and latest Titanium protocol. All three sources of error lead to inflation of diversity estimates. In particular, chimera formation has a hitherto unrealised importance which varies according to amplification protocol. We show that AmpliconNoise allows accurate estimates of OTU number. Just as importantly AmpliconNoise generates the right OTUs even at low sequence differences. We demonstrate that Perseus has very high sensitivity, able to find 99% of chimeras, which is critical when these are present at high

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

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

2016-01-01

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

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

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Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

2017-11-14

The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

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Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

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Minou Nowrousian

2010-04-01

Full Text Available Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data

Phylogenetic analysis of the core histone doublet and DNA topo II genes of Marseilleviridae: evidence of proto-eukaryotic provenance.

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Erives, Albert J

2017-11-28

While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like "doublets" in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of

Roadmap for annotating transposable elements in eukaryote genomes.

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Permal, Emmanuelle; Flutre, Timothée; Quesneville, Hadi

2012-01-01

Current high-throughput techniques have made it feasible to sequence even the genomes of non-model organisms. However, the annotation process now represents a bottleneck to genome analysis, especially when dealing with transposable elements (TE). Combined approaches, using both de novo and knowledge-based methods to detect TEs, are likely to produce reasonably comprehensive and sensitive results. This chapter provides a roadmap for researchers involved in genome projects to address this issue. At each step of the TE annotation process, from the identification of TE families to the annotation of TE copies, we outline the tools and good practices to be used.

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.

Massive expansion of the calpain gene family in unicellular eukaryotes.

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Zhao, Sen; Liang, Zhe; Demko, Viktor; Wilson, Robert; Johansen, Wenche; Olsen, Odd-Arne; Shalchian-Tabrizi, Kamran

2012-09-29

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. 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. 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.

Ensembl Genomes 2013: scaling up access to genome-wide data.

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Kersey, Paul Julian; Allen, James E; Christensen, Mikkel; Davis, Paul; Falin, Lee J; Grabmueller, Christoph; Hughes, Daniel Seth Toney; Humphrey, Jay; Kerhornou, Arnaud; Khobova, Julia; Langridge, Nicholas; McDowall, Mark D; Maheswari, Uma; Maslen, Gareth; Nuhn, Michael; Ong, Chuang Kee; Paulini, Michael; Pedro, Helder; Toneva, Iliana; Tuli, Mary Ann; Walts, Brandon; Williams, Gareth; Wilson, Derek; Youens-Clark, Ken; Monaco, Marcela K; Stein, Joshua; Wei, Xuehong; Ware, Doreen; Bolser, Daniel M; Howe, Kevin Lee; Kulesha, Eugene; Lawson, Daniel; Staines, Daniel Michael

2014-01-01

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species. The project exploits and extends technologies for genome annotation, analysis and dissemination, developed in the context of the vertebrate-focused Ensembl project, and provides a complementary set of resources for non-vertebrate species through a consistent set of programmatic and interactive interfaces. These provide access to data including reference sequence, gene models, transcriptional data, polymorphisms and comparative analysis. This article provides an update to the previous publications about the resource, with a focus on recent developments. These include the addition of important new genomes (and related data sets) including crop plants, vectors of human disease and eukaryotic pathogens. In addition, the resource has scaled up its representation of bacterial genomes, and now includes the genomes of over 9000 bacteria. Specific extensions to the web and programmatic interfaces have been developed to support users in navigating these large data sets. Looking forward, analytic tools to allow targeted selection of data for visualization and download are likely to become increasingly important in future as the number of available genomes increases within all domains of life, and some of the challenges faced in representing bacterial data are likely to become commonplace for eukaryotes in future.

Automatic generation of gene finders for eukaryotic species

DEFF Research Database (Denmark)

Terkelsen, Kasper Munch; Krogh, A.

2006-01-01

and quality of reliable gene annotation grows. Results We present a procedure, Agene, that automatically generates a species-specific gene predictor from a set of reliable mRNA sequences and a genome. We apply a Hidden Markov model (HMM) that implements explicit length distribution modelling for all gene......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...... structure blocks using acyclic discrete phase type distributions. The state structure of the each HMM is generated dynamically from an array of sub-models to include only gene features represented in the training set. Conclusion Acyclic discrete phase type distributions are well suited to model sequence...

Genetic exchange in eukaryotes through horizontal transfer: connected by the mobilome.

Science.gov (United States)

Wallau, Gabriel Luz; Vieira, Cristina; Loreto, Élgion Lúcio Silva

2018-01-01

All living species contain genetic information that was once shared by their common ancestor. DNA is being inherited through generations by vertical transmission (VT) from parents to offspring and from ancestor to descendant species. This process was considered the sole pathway by which biological entities exchange inheritable information. However, Horizontal Transfer (HT), the exchange of genetic information by other means than parents to offspring, was discovered in prokaryotes along with strong evidence showing that it is a very important process by which prokaryotes acquire new genes. For some time now, it has been a scientific consensus that HT events were rare and non-relevant for evolution of eukaryotic species, but there is growing evidence supporting that HT is an important and frequent phenomenon in eukaryotes as well. Here, we will discuss the latest findings regarding HT among eukaryotes, mainly HT of transposons (HTT), establishing HTT once and for all as an important phenomenon that should be taken into consideration to fully understand eukaryotes genome evolution. In addition, we will discuss the latest development methods to detect such events in a broader scale and highlight the new approaches which should be pursued by researchers to fill the knowledge gaps regarding HTT among eukaryotes.

Potential and pitfalls of eukaryotic metagenome skimming: a test case for lichens.

Science.gov (United States)

Greshake, Bastian; Zehr, Simonida; Dal Grande, Francesco; Meiser, Anjuli; Schmitt, Imke; Ebersberger, Ingo

2016-03-01

Whole-genome shotgun sequencing of multispecies communities using only a single library layout is commonly used to assess taxonomic and functional diversity of microbial assemblages. Here, we investigate to what extent such metagenome skimming approaches are applicable for in-depth genomic characterizations of eukaryotic communities, for example lichens. We address how to best assemble a particular eukaryotic metagenome skimming data, what pitfalls can occur, and what genome quality can be expected from these data. To facilitate a project-specific benchmarking, we introduce the concept of twin sets, simulated data resembling the outcome of a particular metagenome sequencing study. We show that the quality of genome reconstructions depends essentially on assembler choice. Individual tools, including the metagenome assemblers Omega and MetaVelvet, are surprisingly sensitive to low and uneven coverages. In combination with the routine of assembly parameter choice to optimize the assembly N50 size, these tools can preclude an entire genome from the assembly. In contrast, MIRA, an all-purpose overlap assembler, and SPAdes, a multisized de Bruijn graph assembler, facilitate a comprehensive view on the individual genomes across a wide range of coverage ratios. Testing assemblers on a real-world metagenome skimming data from the lichen Lasallia pustulata demonstrates the applicability of twin sets for guiding method selection. Furthermore, it reveals that the assembly outcome for the photobiont Trebouxia sp. falls behind the a priori expectation given the simulations. Although the underlying reasons remain still unclear, this highlights that further studies on this organism require special attention during sequence data generation and downstream analysis. © 2015 John Wiley & Sons Ltd.

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.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Science.gov (United States)

Brawley, Susan H; Blouin, Nicolas A; Ficko-Blean, Elizabeth; Wheeler, Glen L; Lohr, Martin; Goodson, Holly V; Jenkins, Jerry W; Blaby-Haas, Crysten E; Helliwell, Katherine E; Chan, Cheong Xin; Marriage, Tara N; Bhattacharya, Debashish; Klein, Anita S; Badis, Yacine; Brodie, Juliet; Cao, Yuanyu; Collén, Jonas; Dittami, Simon M; Gachon, Claire M M; Green, Beverley R; Karpowicz, Steven J; Kim, Jay W; Kudahl, Ulrich Johan; Lin, Senjie; Michel, Gurvan; Mittag, Maria; Olson, Bradley J S C; Pangilinan, Jasmyn L; Peng, Yi; Qiu, Huan; Shu, Shengqiang; Singer, John T; Smith, Alison G; Sprecher, Brittany N; Wagner, Volker; Wang, Wenfei; Wang, Zhi-Yong; Yan, Juying; Yarish, Charles; Zäuner-Riek, Simone; Zhuang, Yunyun; Zou, Yong; Lindquist, Erika A; Grimwood, Jane; Barry, Kerrie W; Rokhsar, Daniel S; Schmutz, Jeremy; Stiller, John W; Grossman, Arthur R; Prochnik, Simon E

2017-08-01

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra , lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution.

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Ajeet Mandal

Full Text Available The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro and/or PPG (Pro-Pro-Gly-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids, only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution.

Clusters of orthologous genes for 41 archaeal genomes and implications for evolutionary genomics of archaea

OpenAIRE

Wolf Yuri I; Novichkov Pavel S; Sorokin Alexander V; Makarova Kira S; Koonin Eugene V

2007-01-01

Abstract Background An evolutionary classification of genes from sequenced genomes that distinguishes between orthologs and paralogs is indispensable for genome annotation and evolutionary reconstruction. Shortly after multiple genome sequences of bacteria, archaea, and unicellular eukaryotes became available, an attempt on such a classification was implemented in Clusters of Orthologous Groups of proteins (COGs). Rapid accumulation of genome sequences creates opportunities for refining COGs ...

Genome sequencing of bacteria: sequencing, de novo assembly and rapid analysis using open source tools.

Science.gov (United States)

Kisand, Veljo; Lettieri, Teresa

2013-04-01

De novo genome sequencing of previously uncharacterized microorganisms has the potential to open up new frontiers in microbial genomics by providing insight into both functional capabilities and biodiversity. Until recently, Roche 454 pyrosequencing was the NGS method of choice for de novo assembly because it generates hundreds of thousands of long reads (tools for processing NGS data are increasingly free and open source and are often adopted for both their high quality and role in promoting academic freedom. The error rate of pyrosequencing the Alcanivorax borkumensis genome was such that thousands of insertions and deletions were artificially introduced into the finished genome. Despite a high coverage (~30 fold), it did not allow the reference genome to be fully mapped. Reads from regions with errors had low quality, low coverage, or were missing. The main defect of the reference mapping was the introduction of artificial indels into contigs through lower than 100% consensus and distracting gene calling due to artificial stop codons. No assembler was able to perform de novo assembly comparable to reference mapping. Automated annotation tools performed similarly on reference mapped and de novo draft genomes, and annotated most CDSs in the de novo assembled draft genomes. Free and open source software (FOSS) tools for assembly and annotation of NGS data are being developed rapidly to provide accurate results with less computational effort. Usability is not high priority and these tools currently do not allow the data to be processed without manual intervention. Despite this, genome assemblers now readily assemble medium short reads into long contigs (>97-98% genome coverage). A notable gap in pyrosequencing technology is the quality of base pair calling and conflicting base pairs between single reads at the same nucleotide position. Regardless, using draft whole genomes that are not finished and remain fragmented into tens of contigs allows one to characterize

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

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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

A Collection of Algal Genomes from the JGI

Energy Technology Data Exchange (ETDEWEB)

Kuo, Alan; Grigoriev, Igor

2012-03-19

Algae, defined as photosynthetic eukaryotes other than plants, constitute a major component of fundamental eukaryotic diversity. Acquisition of the ability to conduct oxygenic photosynthesis through endosymbiotic events has been a principal driver of eukaryotic evolution, and today algae continue to underpin aquatic food chains as primary producers. Algae play profound roles in the carbon cycle, can impose health and economic costs through toxic blooms, and are candidate sources for bio-fuels; all of these research areas are part of the mission of DOE?s Joint Genome Institute (JGI). A collection of algal projects ongoing at JGI contributes to each of these areas and illustrates analyses employed in their genome exploration.

A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes

DEFF Research Database (Denmark)

Andersen, Gorm; Bjornberg, Olof; Polakova, Silvia

2008-01-01

Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyv...... of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.......Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces......, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1...

Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

DEFF Research Database (Denmark)

Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

1997-01-01

We have developed a new method for the identification of signal peptides and their cleavage based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome...

Eukaryotic community diversity and spatial variation during drinking water production (by seawater desalination) and distribution in a full-scale network

KAUST Repository

Belila, Abdelaziz

2016-12-01

Eukaryotic microorganisms are naturally present in many water resources and can enter, grow and colonize water treatment and transport systems, including reservoirs, pipes and premise plumbing. In this study, we explored the eukaryotic microbial community structure in water during the (i) production of drinking water in a seawater desalination plant and (ii) transport of the drinking water in the distribution network. The desalination plant treatment involved pre-treatment (e.g. spruce filters), reverse osmosis (RO) membrane filtration and post-treatment steps (e.g. remineralization). 454 pyrosequencing analysis of the 18S rRNA gene revealed a highly diverse (35 phyla) and spatially variable eukaryotic community during water treatment and distribution. The desalination plant feed water contained a typical marine picoeukaryotic community dominated by Stramenopiles, Alveolates and Porifera. In the desalination plant Ascomycota was the most dominant phylum (15.5% relative abundance), followed by Alveolata (11.9%), unclassified fungi clade (10.9%) and Porifera (10.7%). In the drinking water distribution network, an uncultured fungi phylum was the major group (44.0%), followed by Chordata (17.0%), Ascomycota (11.0%) and Arthropoda (8.0%). Fungi constituted 40% of the total eukaryotic community in the treatment plant and the distribution network and their taxonomic composition was dominated by an uncultured fungi clade (55%). Comparing the plant effluent to the network samples, 84 OTUs (2.1%) formed the core eukaryotic community while 35 (8.4%) and 299 (71.5%) constituted unique OTUs in the produced water at the plant and combined tap water samples from the network, respectively. RO membrane filtration treatment significantly changed the water eukaryotic community composition and structure, highlighting the fact that (i) RO produced water is not sterile and (ii) the microbial community in the final tap water is influenced by the downstream distribution system. The study

[Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

Science.gov (United States)

Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

2012-05-01

To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, PNested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, Pnested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

DEFF Research Database (Denmark)

Nielsen, Olaf; Løbner-Olesen, Anders

2008-01-01

DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells...... prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism...

Hybrid and rogue kinases encoded in the genomes of model eukaryotes.

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Ramaswamy Rakshambikai

Full Text Available The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes-S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens-and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.

Distinguishing friends, foes, and freeloaders in giant genomes.

Science.gov (United States)

Bennetzen, Jeffrey L; Park, Minkyu

2018-04-01

Most annotations of large eukaryotic genomes initially find transposable elements (TEs) and other repeats, then mask them so that subsequent efforts can be concentrated on the annotation and study of non-TE genes. However, TEs often contribute to host biology, and their community biologies are of intrinsic interest. This review discusses the challenges, rationale and technologies for comprehensive TE annotation in the commonly giant genomes of animals and plants. Complete discovery of the TEs in a fully sequenced genome is laborious, but feasible, with current strategies in the hands of a careful researcher. These deep TE studies have begun to provide important perspectives on how genomes evolve and the degree to which genome changes do and do not affect eukaryotic biology. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multiple roles of genome-attached bacteriophage terminal proteins

International Nuclear Information System (INIS)

Redrejo-Rodríguez, Modesto; Salas, Margarita

2014-01-01

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer

Multiple roles of genome-attached bacteriophage terminal proteins

Energy Technology Data Exchange (ETDEWEB)

Redrejo-Rodríguez, Modesto; Salas, Margarita, E-mail: msalas@cbm.csic.es

2014-11-15

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.

Evidence of ancient genome reduction in red algae (Rhodophyta).

Science.gov (United States)

Qiu, Huan; Price, Dana C; Yang, Eun Chan; Yoon, Hwan Su; Bhattacharya, Debashish

2015-08-01

Red algae (Rhodophyta) comprise a monophyletic eukaryotic lineage of ~6,500 species with a fossil record that extends back 1.2 billion years. A surprising aspect of red algal evolution is that sequenced genomes encode a relatively limited gene inventory (~5-10 thousand genes) when compared with other free-living algae or to other eukaryotes. This suggests that the common ancestor of red algae may have undergone extensive genome reduction, which can result from lineage specialization to a symbiotic or parasitic lifestyle or adaptation to an extreme or oligotrophic environment. We gathered genome and transcriptome data from a total of 14 red algal genera that represent the major branches of this phylum to study genome evolution in Rhodophyta. Analysis of orthologous gene gains and losses identifies two putative major phases of genome reduction: (i) in the stem lineage leading to all red algae resulting in the loss of major functions such as flagellae and basal bodies, the glycosyl-phosphatidylinositol anchor biosynthesis pathway, and the autophagy regulation pathway; and (ii) in the common ancestor of the extremophilic Cyanidiophytina. Red algal genomes are also characterized by the recruitment of hundreds of bacterial genes through horizontal gene transfer that have taken on multiple functions in shared pathways and have replaced eukaryotic gene homologs. Our results suggest that Rhodophyta may trace their origin to a gene depauperate ancestor. Unlike plants, it appears that a limited gene inventory is sufficient to support the diversification of a major eukaryote lineage that possesses sophisticated multicellular reproductive structures and an elaborate triphasic sexual cycle. © 2015 Phycological Society of America.

Analysis of high-throughput sequencing and annotation strategies for phage genomes.

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Matthew R Henn

Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes

Science.gov (United States)

Santini, Sebastien; Jeudy, Sandra; Bartoli, Julia; Poirot, Olivier; Lescot, Magali; Abergel, Chantal; Barbe, Valérie; Wommack, K. Eric; Noordeloos, Anna A. M.; Brussaard, Corina P. D.; Claverie, Jean-Michel

2013-01-01

Large dsDNA viruses are involved in the population control of many globally distributed species of eukaryotic phytoplankton and have a prominent role in bloom termination. The genus Phaeocystis (Haptophyta, Prymnesiophyceae) includes several high-biomass-forming phytoplankton species, such as Phaeocystis globosa, the blooms of which occur mostly in the coastal zone of the North Atlantic and the North Sea. Here, we report the 459,984-bp-long genome sequence of P. globosa virus strain PgV-16T, encoding 434 proteins and eight tRNAs and, thus, the largest fully sequenced genome to date among viruses infecting algae. Surprisingly, PgV-16T exhibits no phylogenetic affinity with other viruses infecting microalgae (e.g., phycodnaviruses), including those infecting Emiliania huxleyi, another ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to an emerging clade (the Megaviridae) clustering the viruses endowed with the largest known genomes, including Megavirus, Mimivirus (both infecting acanthamoeba), and a virus infecting the marine microflagellate grazer Cafeteria roenbergensis. Seventy-five percent of the best matches of PgV-16T–predicted proteins correspond to two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from a hypersaline lake in Antarctica (Organic Lake), the hosts of which are unknown. As for OLPVs and other Megaviridae, the PgV-16T sequence data revealed the presence of a virophage-like genome. However, no virophage particle was detected in infected P. globosa cultures. The presence of many genes found only in Megaviridae in its genome and the presence of an associated virophage strongly suggest that PgV-16T shares a common ancestry with the largest known dsDNA viruses, the host range of which already encompasses the earliest diverging branches of domain Eukarya. PMID:23754393

Identification and analysis of common bean (Phaseolus vulgaris L. transcriptomes by massively parallel pyrosequencing

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Thimmapuram Jyothi

2011-10-01

Full Text Available Abstract Background Common bean (Phaseolus vulgaris is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptome analysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. Results We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt. These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC end sequences, and a total of 21% of the unigenes (12,724 including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs and transcription factors were also identified in this study. Conclusions This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and

Recognition of extremophilic archaeal viruses by eukaryotic cells

DEFF Research Database (Denmark)

Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur

2016-01-01

followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path......Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration...

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. © 2014 Elsevier Inc. All rights reserved.

Genomic evolution of 11 type strains within family Planctomycetaceae.

Directory of Open Access Journals (Sweden)

Min Guo

Full Text Available The species in family Planctomycetaceae are ideal groups for investigating the origin of eukaryotes. Their cells are divided by a lipidic intracytoplasmic membrane and they share a number of eukaryote-like molecular characteristics. However, their genomic structures, potential abilities, and evolutionary status are still unknown. In this study, we searched for common protein families and a core genome/pan genome based on 11 sequenced species in family Planctomycetaceae. Then, we constructed phylogenetic tree based on their 832 common protein families. We also annotated the 11 genomes using the Clusters of Orthologous Groups database. Moreover, we predicted and reconstructed their core/pan metabolic pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes orthology system. Subsequently, we identified genomic islands (GIs and structural variations (SVs among the five complete genomes and we specifically investigated the integration of two Planctomycetaceae plasmids in all 11 genomes. The results indicate that Planctomycetaceae species share diverse genomic variations and unique genomic characteristics, as well as have huge potential for human applications.

Requirements and standards for organelle genome databases

Energy Technology Data Exchange (ETDEWEB)

Boore, Jeffrey L.

2006-01-09

Mitochondria and plastids (collectively called organelles)descended from prokaryotes that adopted an intracellular, endosymbioticlifestyle within early eukaryotes. Comparisons of their remnant genomesaddress a wide variety of biological questions, especially when includingthe genomes of their prokaryotic relatives and the many genes transferredto the eukaryotic nucleus during the transitions from endosymbiont toorganelle. The pace of producing complete organellar genome sequences nowmakes it unfeasible to do broad comparisons using the primary literatureand, even if it were feasible, it is now becoming uncommon for journalsto accept detailed descriptions of genome-level features. Unfortunatelyno database is currently useful for this task, since they have littlestandardization and are riddled with error. Here I outline what iscurrently wrong and what must be done to make this data useful to thescientific community.

Metabarcoding Analysis of Phytophthora Diversity Using Genus-Specific Primers and 454 Pyrosequencing.

Science.gov (United States)

Prigigallo, Maria I; Abdelfattah, Ahmed; Cacciola, Santa O; Faedda, Roberto; Sanzani, Simona M; Cooke, David E L; Schena, L

2016-03-01

A metabarcoding method based on genus-specific primers and 454 pyrosequencing was utilized to investigate the genetic diversity of Phytophthora spp. in soil and root samples of potted plants, from eight nurseries. Pyrosequencing enabled the detection of 25 Phytophthora phylotypes distributed in seven different clades and provided a much higher resolution than a corresponding cloning/Sanger sequencing approach. Eleven of these phylotypes, including P. cactorum, P. citricola s.str., P. palmivora, P. palmivora-like, P. megasperma or P. gonapodyides, P. ramorum, and five putative new Phytophthora species phylogenetically related to clades 1, 2, 4, 6, and 7 were detected only with the 454 pyrosequencing approach. We also found an additional 18 novel records of a phylotype in a particular nursery that were not detected with cloning/Sanger sequencing. Several aspects confirmed the reliability of the method: (i) many identical sequence types were identified independently in different nurseries, (ii) most sequence types identified with 454 pyrosequencing were identical to those from the cloning/Sanger sequencing approach and/or perfectly matched GenBank deposited sequences, and (iii) the divergence noted between sequence types of putative new Phytophthora species and all other detected sequences was sufficient to rule out sequencing errors. The proposed method represents a powerful tool to study Phytophthora diversity providing that particular attention is paid to the analysis of 454 pyrosequencing raw read sequences and to the identification of sequence types.

Regulated eukaryotic DNA replication origin firing with purified proteins.

Science.gov (United States)

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Comparative radiobiology of genetic loci of eukaryots as the basis of the general theory of mutations

International Nuclear Information System (INIS)

Aleksandrov, I.D.

1983-01-01

One of the fundamental problems of modern molecular cellular radiobiology is to reveal general and peculiar processes of the formation of gene mutations and chromosome aberrations in each stage of their formation in the irradiated genome of the higher eukaryots. The solution of the problems depends on the development of research within the framework of comparative radiobiology of genetic loci of the higher eukaryots that makes it possible to study quantitative regularities in the formation of gene (point) mutations and chromosome aberrations in one object and in the same experiment

The genome of the obligate intracellular parasite Trachipleistophora hominis: new insights into microsporidian genome dynamics and reductive evolution.

Directory of Open Access Journals (Sweden)

Eva Heinz

Full Text Available The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome

Searching for genomic constraints

Energy Technology Data Exchange (ETDEWEB)

Lio` , P [Cambridge, Univ. (United Kingdom). Genetics Dept.; Ruffo, S [Florence, Univ. (Italy). Fac. di Ingegneria. Dipt. di Energetica ` S. Stecco`

1998-01-01

The authors have analyzed general properties of very long DNA sequences belonging to simple and complex organisms, by using different correlation methods. They have distinguished those base compositional rules that concern the entire genome which they call `genomic constraints` from the rules that depend on the `external natural selection` acting on single genes, i. e. protein-centered constraints. They show that G + C content, purine / pyrimidine distributions and biological complexity of the organism are the most important factors which determine base compositional rules and genome complexity. Three main facts are here reported: bacteria with high G + C content have more restrictions on base composition than those with low G + C content; at constant G + C content more complex organisms, ranging from prokaryotes to higher eukaryotes (e.g. human) display an increase of repeats 10-20 nucleotides long, which are also partly responsible for long-range correlations; work selection of length 3 to 10 is stronger in human and in bacteria for two distinct reasons. With respect to previous studies, they have also compared the genomic sequence of the archeon Methanococcus jannaschii with those of bacteria and eukaryotes: it shows sometimes an intermediate statistical behaviour.

Searching for genomic constraints

International Nuclear Information System (INIS)

Lio', P.; Ruffo, S.

1998-01-01

The authors have analyzed general properties of very long DNA sequences belonging to simple and complex organisms, by using different correlation methods. They have distinguished those base compositional rules that concern the entire genome which they call 'genomic constraints' from the rules that depend on the 'external natural selection' acting on single genes, i. e. protein-centered constraints. They show that G + C content, purine / pyrimidine distributions and biological complexity of the organism are the most important factors which determine base compositional rules and genome complexity. Three main facts are here reported: bacteria with high G + C content have more restrictions on base composition than those with low G + C content; at constant G + C content more complex organisms, ranging from prokaryotes to higher eukaryotes (e.g. human) display an increase of repeats 10-20 nucleotides long, which are also partly responsible for long-range correlations; work selection of length 3 to 10 is stronger in human and in bacteria for two distinct reasons. With respect to previous studies, they have also compared the genomic sequence of the archeon Methanococcus jannaschii with those of bacteria and eukaryotes: it shows sometimes an intermediate statistical behaviour

Barcoding lichen-forming fungi using 454 pyrosequencing is challenged by artifactual and biological sequence variation.

Science.gov (United States)

Mark, Kristiina; Cornejo, Carolina; Keller, Christine; Flück, Daniela; Scheidegger, Christoph

2016-09-01

Although lichens (lichen-forming fungi) play an important role in the ecological integrity of many vulnerable landscapes, only a minority of lichen-forming fungi have been barcoded out of the currently accepted ∼18 000 species. Regular Sanger sequencing can be problematic when analyzing lichens since saprophytic, endophytic, and parasitic fungi live intimately admixed, resulting in low-quality sequencing reads. Here, high-throughput, long-read 454 pyrosequencing in a GS FLX+ System was tested to barcode the fungal partner of 100 epiphytic lichen species from Switzerland using fungal-specific primers when amplifying the full internal transcribed spacer region (ITS). The present study shows the potential of DNA barcoding using pyrosequencing, in that the expected lichen fungus was successfully sequenced for all samples except one. Alignment solutions such as BLAST were found to be largely adequate for the generated long reads. In addition, the NCBI nucleotide database-currently the most complete database for lichen-forming fungi-can be used as a reference database when identifying common species, since the majority of analyzed lichens were identified correctly to the species or at least to the genus level. However, several issues were encountered, including a high sequencing error rate, multiple ITS versions in a genome (incomplete concerted evolution), and in some samples the presence of mixed lichen-forming fungi (possible lichen chimeras).

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Science.gov (United States)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

NARCIS (Netherlands)

Procopio, Luciano; Alvarez, Vanessa M.; Jurelevicius, Diogo A.; Hansen, Lars; Sorensen, Soren J.; Cardoso, Janine S.; Padula, Marcelo; Leitao, Alvaro C.; Seldin, Lucy; van Elsas, Jan Dirk

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average

Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle

KAUST Repository

Aranda, Manuel

2016-12-22

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle

KAUST Repository

Aranda, Manuel; Li, Yangyang; Liew, Yi Jin; Baumgarten, Sebastian; Simakov, O.; Wilson, M. C.; Piel, J.; Ashoor, Haitham; Bougouffa, Salim; Bajic, Vladimir B.; Ryu, Tae Woo; Ravasi, Timothy; Bayer, Till; Micklem, G.; Kim, H.; Bhak, J.; LaJeunesse, T. C.; Voolstra, Christian R.

2016-01-01

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

Genomic Analysis of Complex Microbial Communities in Wounds

Science.gov (United States)

2012-01-01

Permutation Multivariate Analysis of Variance ( PerMANOVA ). We used PerMANOVA to test the null-hypothesis of no... permutation -based version of the multivariate analysis of variance (MANOVA). PerMANOVA uses the distances between samples to partition variance and...coli. Antibiotics, bacteria, community analysis , diabetes, pyrosequencing, wound, wound therapy, 16S rRNA gene Genomic Analysis of Complex

Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity

Energy Technology Data Exchange (ETDEWEB)

Parfrey, Laura Wegener; Walters, William A.; Lauber, Christian L.; Clemente, Jose C.; Berg-Lyons, Donna; Teiling, Clotilde; Kodira, Chinnappa; Mohiuddin, Mohammed; Brunelle, Julie; Driscoll, Mark; Fierer, Noah; Gilbert, Jack A.; Knight, Rob

2014-06-19

Eukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

Science.gov (United States)

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

Energy Technology Data Exchange (ETDEWEB)

Bomberg, M.; Nyyssoenen, M.; Itaevaara, M. [VTT Technical Research Centre of Finland, Espoo (Finland)

2012-06-15

Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.

Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

Energy Technology Data Exchange (ETDEWEB)

Bomberg, M; Nyyssoenen, M; Itaevaara, M [VTT Technical Research Centre of Finland, Espoo (Finland)

2012-06-15

Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.

Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

International Nuclear Information System (INIS)

Bomberg, M.; Nyyssoenen, M.; Itaevaara, M.

2012-06-01

Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected

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.

Susceptibilities to DNA Structural Transitions within Eukaryotic Genomes

Science.gov (United States)

Zhabinskaya, Dina; Benham, Craig; Madden, Sally

2012-02-01

We analyze the competitive transitions to alternate secondary DNA structures in a negatively supercoiled DNA molecule of kilobase length and specified base sequence. We use statistical mechanics to calculate the competition among all regions within the sequence that are susceptible to transitions to alternate structures. We use an approximate numerical method since the calculation of an exact partition function is numerically cumbersome for DNA molecules of lengths longer than hundreds of base pairs. This method yields accurate results in reasonable computational times. We implement algorithms that calculate the competition between transitions to denatured states and to Z-form DNA. We analyze these transitions near the transcription start sites (TSS) of a set of eukaryotic genes. We find an enhancement of Z-forming regions upstream of the TSS and a depletion of denatured regions around the start sites. We confirm that these finding are statistically significant by comparing our results to a set of randomized genes with preserved base composition at each position relative to the gene start sites. When we study the correlation of these transitions in orthologous mouse and human genes we find a clear evolutionary conservation of both types of transitions around the TSS.

Cultivation of seaweed Gracilaria lemaneiformis enhanced biodiversity in a eukaryotic plankton community as revealed via metagenomic analyses.

Science.gov (United States)

Chai, Zhao Yang; He, Zhi Li; Deng, Yun Yan; Yang, Yu Feng; Tang, Ying Zhong

2018-02-01

Plankton diversity reflects the quality and health of waters and should be monitored as a critical feature of marine ecosystems. This study applied a pair of 28S rRNA gene-specific primers and pyrosequencing to assess the effects of large-scale cultivation of the seaweed Gracilaria lemaneiformis on the biodiversity of eukaryotic plankton community in the coastal water of Guangdong, China. With 1 million sequences (2,221 operational taxonomic units [OTUs]) obtained from 51 samples, we found that the biodiversity of eukaryotic plankton community was significantly higher in the seaweed cultivation area than that in the nearby control area as reflected in OTU richness, evenness (Shannon-Wiener index) and dominance (Simpson index) for total plankton community and its four subcategories when Gracilaria biomass reached the maximum, while no such a significant difference was observed before seaweed inoculation. Our laboratory experiment using an artificial phytoplankton community of nine species observed the same effects of Gracilaria exposure. Principal component analysis and principal coordinates analysis showed the plankton community structure in cultivation area markedly differed from the control area when Gracilaria biomass reached its maximum. Redundancy analysis showed that G. lemaneiformis was the critical factor in controlling the dynamics of eukaryotic plankton communities in the studied coastal ecosystem. Our results explicitly demonstrated G. lemaneiformis cultivation could enhance biodiversity of plankton community via allelopathy, which prevents one or several plankton species from blooming and consequently maintains a relatively higher biodiversity. Our study provided further support for using large-scale G. lemaneiformis cultivation as an effective approach for improving costal ecosystem health. © 2018 John Wiley & Sons Ltd.

Comparative genomic analysis of multi-subunit tethering complexes demonstrates an ancient pan-eukaryotic complement and sculpting in Apicomplexa.

Directory of Open Access Journals (Sweden)

Christen M Klinger

Full Text Available Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs, factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardiatheta and Bigelowiellanatans. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during

Genomic minimalism in the early diverging intestinal parasite Giardia lamblia.

Science.gov (United States)

Morrison, Hilary G; McArthur, Andrew G; Gillin, Frances D; Aley, Stephen B; Adam, Rodney D; Olsen, Gary J; Best, Aaron A; Cande, W Zacheus; Chen, Feng; Cipriano, Michael J; Davids, Barbara J; Dawson, Scott C; Elmendorf, Heidi G; Hehl, Adrian B; Holder, Michael E; Huse, Susan M; Kim, Ulandt U; Lasek-Nesselquist, Erica; Manning, Gerard; Nigam, Anuranjini; Nixon, Julie E J; Palm, Daniel; Passamaneck, Nora E; Prabhu, Anjali; Reich, Claudia I; Reiner, David S; Samuelson, John; Svard, Staffan G; Sogin, Mitchell L

2007-09-28

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Systematics of Short-range Correlations in Eukaryotic Genomes

Science.gov (United States)

Hameister, Jörn; Helm, Werner E.; Hütt, Marc-Thorsten; Dehnert, Manuel

Attempts to identify a species on the basis of its DNA sequence on purely statistical grounds have been formulated for more than a decade. Solving this problem could have a huge impact on understanding processes of genome evolution and on the design of classification schemes for DNA sequences.

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones ▿

Science.gov (United States)

Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.

2009-01-01

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642

Phylogenetic distribution of large-scale genome patchiness

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Hackenberg Michael

2008-04-01

Full Text Available Abstract Background The phylogenetic distribution of large-scale genome structure (i.e. mosaic compositional patchiness has been explored mainly by analytical ultracentrifugation of bulk DNA. However, with the availability of large, good-quality chromosome sequences, and the recently developed computational methods to directly analyze patchiness on the genome sequence, an evolutionary comparative analysis can be carried out at the sequence level. Results The local variations in the scaling exponent of the Detrended Fluctuation Analysis are used here to analyze large-scale genome structure and directly uncover the characteristic scales present in genome sequences. Furthermore, through shuffling experiments of selected genome regions, computationally-identified, isochore-like regions were identified as the biological source for the uncovered large-scale genome structure. The phylogenetic distribution of short- and large-scale patchiness was determined in the best-sequenced genome assemblies from eleven eukaryotic genomes: mammals (Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, and Canis familiaris, birds (Gallus gallus, fishes (Danio rerio, invertebrates (Drosophila melanogaster and Caenorhabditis elegans, plants (Arabidopsis thaliana and yeasts (Saccharomyces cerevisiae. We found large-scale patchiness of genome structure, associated with in silico determined, isochore-like regions, throughout this wide phylogenetic range. Conclusion Large-scale genome structure is detected by directly analyzing DNA sequences in a wide range of eukaryotic chromosome sequences, from human to yeast. In all these genomes, large-scale patchiness can be associated with the isochore-like regions, as directly detected in silico at the sequence level.

Characterization of an eukaryotic peptide deformylase from Plasmodium falciparum.

Science.gov (United States)

Bracchi-Ricard, V; Nguyen, K T; Zhou, Y; Rajagopalan, P T; Chakrabarti, D; Pei, D

2001-12-15

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Until recently, PDF has been thought as an enzyme unique to the bacterial kingdom. Searches of the genomic DNA databases identified several genes that encode proteins of high sequence homology to bacterial PDF from eukaryotic organisms. The cDNA encoding Plasmodium falciparum PDF (PfPDF) has been cloned and overexpressed in Escherichia coli. The recombinant protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is inhibited by specific PDF inhibitors. Western blot analysis indicated expression of mature PfPDF in trophozoite, schizont, and segmenter stages of intraerythrocytic development. These results provide strong evidence that a functional PDF is present in P. falciparum. In addition, PDF inhibitors inhibited the growth of P. falciparum in the intraerythrocytic culture. (c)2001 Elsevier Science.

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.

Insights into the Initiation of Eukaryotic DNA Replication.

Science.gov (United States)

Bruck, Irina; Perez-Arnaiz, Patricia; Colbert, Max K; Kaplan, Daniel L

2015-01-01

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution.

Science.gov (United States)

Rogozin, Igor B; Wolf, Yuri I; Sorokin, Alexander V; Mirkin, Boris G; Koonin, Eugene V

2003-09-02

Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.

i-Genome: A database to summarize oligonucleotide data in genomes

Directory of Open Access Journals (Sweden)

Chang Yu-Chung

2004-10-01

Full Text Available Abstract Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.

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Jonathan A Eisen

2006-09-01

Full Text Available The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC, which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases, using diverse resources (e.g., proteases and transporters, and generating structural complexity (e.g., kinesins and dyneins. In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates, no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from

Genome-wide analysis of the phosphoinositide kinome from two ciliates reveals novel evolutionary links for phosphoinositide kinases in eukaryotic cells.

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George Leondaritis

Full Text Available BACKGROUND: The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. CONCLUSIONS/SIGNIFICANCE: Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory

Variation in recombination frequency and distribution across eukaryotes: patterns and processes

Science.gov (United States)

Feulner, Philine G. D.; Johnston, Susan E.; Santure, Anna W.; Smadja, Carole M.

2017-01-01

Recombination, the exchange of DNA between maternal and paternal chromosomes during meiosis, is an essential feature of sexual reproduction in nearly all multicellular organisms. While the role of recombination in the evolution of sex has received theoretical and empirical attention, less is known about how recombination rate itself evolves and what influence this has on evolutionary processes within sexually reproducing organisms. Here, we explore the patterns of, and processes governing recombination in eukaryotes. We summarize patterns of variation, integrating current knowledge with an analysis of linkage map data in 353 organisms. We then discuss proximate and ultimate processes governing recombination rate variation and consider how these influence evolutionary processes. Genome-wide recombination rates (cM/Mb) can vary more than tenfold across eukaryotes, and there is large variation in the distribution of recombination events across closely related taxa, populations and individuals. We discuss how variation in rate and distribution relates to genome architecture, genetic and epigenetic mechanisms, sex, environmental perturbations and variable selective pressures. There has been great progress in determining the molecular mechanisms governing recombination, and with the continued development of new modelling and empirical approaches, there is now also great opportunity to further our understanding of how and why recombination rate varies. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109219

Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes

Science.gov (United States)

Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

2015-01-01

Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

Diversity and distribution of eukaryotic microbes in and around a brine pool adjacent to the Thuwal cold seeps in the Red Sea

KAUST Repository

Wang, Yong

2014-02-04

A hypoxic/suboxic brine pool at a depth of about 850 m was discovered near the Thuwal cold seeps in the Red Sea. Filled with high concentrations of hydrogen sulfide and ammonia, such a brine pool might limit the spread of eukaryotic organisms. Here, we compared the communities of the eukaryotic microbes in a microbial mat, sediments and water samples distributed in 7 sites within and adjacent to the brine pool. Taxonomic classification of the pyrosequenced 18S rRNA amplicon reads showed that fungi highly similar to the species identified along the Arabic coast were almost ubiquitous in the water and sediment samples, supporting their wide distribution in various environments. The microbial mat displayed the highest species diversity and contained grazers and a considerable percentage of unclassified species. Phylogeny-based methods revealed novel lineages representing a majority of the reads from the interface between the sea water and brine pool. Phylogenetic relationships with more reference sequences suggest that the lineages were affiliated with novel Alveolata and Euglenozoa inhabiting the interface where chemosynthetic prokaryotes are highly proliferative due to the strong chemocline and halocline. The brine sediments harbored abundant species highly similar to invertebrate gregarine parasites identified in different oxygen-depleted sediments. Therefore, the present findings support the uniqueness of some microbial eukaryotic groups in this cold seep brine system. 2014 Wang, Zhang, Cao, Shek, Tian, Wong, Batang, Al-suwailem and Qian.

insights from the genome of Melitaea cinxia

OpenAIRE

Ahola, Virpi; Wahlberg, Niklas; Frilander, Mikko J.

2017-01-01

The first lepidopteran genome (Bombyx mori) was published in 2004. Ten years later the genome of Melitaea cinxia came out as the third butterfly genome published, and the first eukaryotic genome sequenced in Finland. Owing to Ilkka Hanski, the M. cinxia system in the angstrom land Islands has become a famous model for metapopulation biology. More than 20 years of research on this system provides a strong ecological basis upon which a genetic framework could be built. Genetic knowledge is an e...

Re-evaluating the green versus red signal in eukaryotes with secondary plastid of red algal origin

KAUST Repository

Burki, Fabien

2012-05-16

The transition from endosymbiont to organelle in eukaryotic cells involves the transfer of significant numbers of genes to the host genomes, a process known as endosymbiotic gene transfer (EGT). In the case of plastid organelles, EGTs have been shown to leave a footprint in the nuclear genome that can be indicative of ancient photosynthetic activity in present-day plastid-lacking organisms, or even hint at the existence of cryptic plastids. Here,we evaluated the impact of EGTon eukaryote genomes by reanalyzing the recently published EST dataset for Chromera velia, an interesting test case of a photosynthetic alga closely related to apicomplexan parasites. Previously, 513 genes were reported to originate from red and green algae in a 1:1 ratio. In contrast, by manually inspecting newly generated trees indicating putative algal ancestry, we recovered only 51 genes congruent with EGT, of which 23 and 9 were of red and green algal origin, respectively,whereas 19 were ambiguous regarding the algal provenance.Our approach also uncovered 109 genes that branched within a monocot angiosperm clade, most likely representing a contamination. We emphasize the lack of congruence and the subjectivity resulting from independent phylogenomic screens for EGT, which appear to call for extreme caution when drawing conclusions for major evolutionary events. 2012 The Author(s).

Characterization and Evolution of the Cell Cycle-Associated Mob Domain-Containing Proteins in Eukaryotes

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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.

A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

DEFF Research Database (Denmark)

Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

1997-01-01

We have developed a new method for the identication of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs signicantly better than previous prediction schemes, and can easily be applied to genome...

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

Energy Technology Data Exchange (ETDEWEB)

Merchant, Sabeeha S

2007-04-09

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Long- and short-term selective forces on malaria parasite genomes

DEFF Research Database (Denmark)

Nygaard, Sanne; Braunstein, Alexander; Malsen, Gareth

2010-01-01

Plasmodium parasites, the causal agents of malaria, result in more than 1 million deaths annually. Plasmodium are unicellular eukaryotes with small ~23 Mb genomes encoding ~5200 protein-coding genes. The protein-coding genes comprise about half of these genomes. Although evolutionary processes ha...

Genome-wide survey of repetitive DNA elements in the button mushroom Agaricus bisporus

NARCIS (Netherlands)

Foulongne-Oriol, M.; Murat, C.; Castanera, R.; Ramírez, L.; Sonnenberg, A.S.M.

2013-01-01

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of

Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat.

Science.gov (United States)

Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

2013-10-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. © 2013 Ben-Gurion University The Plant Journal © 2013 John Wiley & Sons Ltd.

The DNA-encoded nucleosome organization of a eukaryotic genome.

Science.gov (United States)

Kaplan, Noam; Moore, Irene K; Fondufe-Mittendorf, Yvonne; Gossett, Andrea J; Tillo, Desiree; Field, Yair; LeProust, Emily M; Hughes, Timothy R; Lieb, Jason D; Widom, Jonathan; Segal, Eran

2009-03-19

Nucleosome organization is critical for gene regulation. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for approximately 40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.

GOBASE: an organelle genome database

OpenAIRE

O?Brien, Emmet A.; Zhang, Yue; Wang, Eric; Marie, Veronique; Badejoko, Wole; Lang, B. Franz; Burger, Gertraud

2008-01-01

The organelle genome database GOBASE, now in its 21st release (June 2008), contains all published mitochondrion-encoded sequences (?913 000) and chloroplast-encoded sequences (?250 000) from a wide range of eukaryotic taxa. For all sequences, information on related genes, exons, introns, gene products and taxonomy is available, as well as selected genome maps and RNA secondary structures. Recent major enhancements to database functionality include: (i) addition of an interface for RNA editing...

Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

Science.gov (United States)

Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

2017-06-02

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Dispersed repetitive sequences in eukaryotic genomes and their possible biological significance

International Nuclear Information System (INIS)

Georgiev, G.P.; Kramerov, D.A.; Ryskov, A.P.; Skryabin, K.G.; Lukanidin, E.M.

1983-01-01

In this paper is described the properties of a novel mouse mdg-like element, the A2 sequence, which is the most abundant repetitive sequence. We also characterized an ubiquitous B2 sequence that represents, after B1, the dominant family among the short interspersed repeats of the mouse genome. The existence of some putative transposition intermediates was shown for repeats of both A and B types of the mouse genome. These are closed circular DNA of the A type and small polyadenylated B + RNAs. The fundamental question that arises is whether these sequences are simply selfish DNA capable of transpositions or do they fulfill some useful biological functions within the genome. 66 references, 11 figures, 1 table

454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides.

Science.gov (United States)

Zagrobelny, Mika; Scheibye-Alsing, Karsten; Jensen, Niels Bjerg; Møller, Birger Lindberg; Gorodkin, Jan; Bak, Søren

2009-12-02

An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects

454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides

Directory of Open Access Journals (Sweden)

Jensen Niels

2009-12-01

Full Text Available Abstract Background An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae. Results Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being

The reduced kinome of Ostreococcus tauri: core eukaryotic signalling components in a tractable model species.

Science.gov (United States)

Hindle, Matthew M; Martin, Sarah F; Noordally, Zeenat B; van Ooijen, Gerben; Barrios-Llerena, Martin E; Simpson, T Ian; Le Bihan, Thierry; Millar, Andrew J

2014-08-02

The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette. Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways - TOR, MAPK, and the circadian clock - we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs. We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes.

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

Science.gov (United States)

Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

2015-06-25

Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex

Microbial analysis in primary and persistent endodontic infections by using pyrosequencing.

Science.gov (United States)

Hong, Bo-Young; Lee, Tae-Kwon; Lim, Sang-Min; Chang, Seok Woo; Park, Joonhong; Han, Seung Hyun; Zhu, Qiang; Safavi, Kamran E; Fouad, Ashraf F; Kum, Kee Yeon

2013-09-01

The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera

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Elsik Chris

2010-10-01

Full Text Available Abstract Background The ectoparasitic mite Varroa destructor has emerged as the primary pest of domestic honey bees (Apis mellifera. Here we present an initial survey of the V. destructor genome carried out to advance our understanding of Varroa biology and to identify new avenues for mite control. This sequence survey provides immediate resources for molecular and population-genetic analyses of Varroa-Apis interactions and defines the challenges ahead for a comprehensive Varroa genome project. Results The genome size was estimated by flow cytometry to be 565 Mbp, larger than most sequenced insects but modest relative to some other Acari. Genomic DNA pooled from ~1,000 mites was sequenced to 4.3× coverage with 454 pyrosequencing. The 2.4 Gbp of sequencing reads were assembled into 184,094 contigs with an N50 of 2,262 bp, totaling 294 Mbp of sequence after filtering. Genic sequences with homology to other eukaryotic genomes were identified on 13,031 of these contigs, totaling 31.3 Mbp. Alignment of protein sequence blocks conserved among V. destructor and four other arthropod genomes indicated a higher level of sequence divergence within this mite lineage relative to the tick Ixodes scapularis. A number of microbes potentially associated with V. destructor were identified in the sequence survey, including ~300 Kbp of sequence deriving from one or more bacterial species of the Actinomycetales. The presence of this bacterium was confirmed in individual mites by PCR assay, but varied significantly by age and sex of mites. Fragments of a novel virus related to the Baculoviridae were also identified in the survey. The rate of single nucleotide polymorphisms (SNPs in the pooled mites was estimated to be 6.2 × 10-5per bp, a low rate consistent with the historical demography and life history of the species. Conclusions This survey has provided general tools for the research community and novel directions for investigating the biology and control of

Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

Science.gov (United States)

Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

2015-01-01

Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing® technology

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Gallagher Margaret L

2009-08-01

Full Text Available Abstract Background Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1–2 are derived from CYP2A7, and exons 3–9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. Methods A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow. Results Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics. Conclusion This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.

Highly variable rates of genome rearrangements between hemiascomycetous yeast lineages.

Directory of Open Access Journals (Sweden)

2006-03-01

Full Text Available Hemiascomycete yeasts cover an evolutionary span comparable to that of the entire phylum of chordates. Since this group currently contains the largest number of complete genome sequences it presents unique opportunities to understand the evolution of genome organization in eukaryotes. We inferred rates of genome instability on all branches of a phylogenetic tree for 11 species and calculated species-specific rates of genome rearrangements. We characterized all inversion events that occurred within synteny blocks between six representatives of the different lineages. We show that the rates of macro- and microrearrangements of gene order are correlated within individual lineages but are highly variable across different lineages. The most unstable genomes correspond to the pathogenic yeasts Candida albicans and Candida glabrata. Chromosomal maps have been intensively shuffled by numerous interchromosomal rearrangements, even between species that have retained a very high physical fraction of their genomes within small synteny blocks. Despite this intensive reshuffling of gene positions, essential genes, which cluster in low recombination regions in the genome of Saccharomyces cerevisiae, tend to remain syntenic during evolution. This work reveals that the high plasticity of eukaryotic genomes results from rearrangement rates that vary between lineages but also at different evolutionary times of a given lineage.

Complete Sequence and Analysis of the Mitochondrial Genome of Hemiselmis andersenii CCMP644 (Cryptophyceae

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Bowman Sharen

2008-05-01

Full Text Available Abstract Background Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote endosymbiosis. Cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. Results The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a ~20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22–336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu gene and possesses a trnS-derived 'trnK(uuu', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher

Genome-wide analysis of tandem repeats in plants and green algae

Science.gov (United States)

Zhixin Zhao; Cheng Guo; Sreeskandarajan Sutharzan; Pei Li; Craig Echt; Jie Zhang; Chun Liang

2014-01-01

Tandem repeats (TRs) extensively exist in the genomes of prokaryotes and eukaryotes. Based on the sequenced genomes and gene annotations of 31 plant and algal species in Phytozome version 8.0 (http://www.phytozome.net/), we examined TRs in a genome-wide scale, characterized their distributions and motif features, and explored their putative biological functions. Among...

Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes

NARCIS (Netherlands)

Revuelta, M.V.; Kan, van J.A.L.; Kay, J.; Have, ten A.

2014-01-01

The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the

A sweetpotato gene index established by de novo assembly of pyrosequencing and Sanger sequences and mining for gene-based microsatellite markers

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Solis Julio

2010-10-01

Full Text Available Abstract Background Sweetpotato (Ipomoea batatas (L. Lam., a hexaploid outcrossing crop, is an important staple and food security crop in developing countries in Africa and Asia. The availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. Previously existing sequence data were restricted to around 22,000 expressed sequence tag (EST sequences and ~ 1,500 GenBank sequences. We have used 454 pyrosequencing to augment the available gene sequence information to enhance functional genomics and marker design for this plant species. Results Two quarter 454 pyrosequencing runs used two normalized cDNA collections from stems and leaves from drought-stressed sweetpotato clone Tanzania and yielded 524,209 reads, which were assembled together with 22,094 publically available expressed sequence tags into 31,685 sets of overlapping DNA segments and 34,733 unassembled sequences. Blastx comparisons with the UniRef100 database allowed annotation of 23,957 contigs and 15,342 singletons resulting in 24,657 putatively unique genes. Further, 27,119 sequences had no match to protein sequences of UniRef100database. On the basis of this gene index, we have identified 1,661 gene-based microsatellite sequences, of which 223 were selected for testing and 195 were successfully amplified in a test panel of 6 hexaploid (I. batatas and 2 diploid (I. trifida accessions. Conclusions The sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous EST assemblies. A searchable version of the gene index, including a blastn function, is available at http://www.cipotato.org/sweetpotato_gene_index.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome

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Modern biological analyses are often assisted by recent technologies making the sequencing of complex genomes both technically possible and feasible. We recently sequenced the tomato genome that, like many eukaryotic genomes, is large and complex. Current sequencing technologies allow the developmen...

Mitigating Mitochondrial Genome Erosion Without Recombination.

Science.gov (United States)

Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

2017-11-01

Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.

Yeast genome sequencing:

DEFF Research Database (Denmark)

Piskur, Jure; Langkjær, Rikke Breinhold

2004-01-01

For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

Insights into structural variations and genome rearrangements in prokaryotic genomes.

Science.gov (United States)

Periwal, Vinita; Scaria, Vinod

2015-01-01

Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expressed Peptide Tags: An additional layer of data for genome annotation

Energy Technology Data Exchange (ETDEWEB)

Savidor, Alon [ORNL; Donahoo, Ryan S [ORNL; Hurtado-Gonzales, Oscar [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Shah, Manesh B [ORNL; Lamour, Kurt H [ORNL; McDonald, W Hayes [ORNL

2006-01-01

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller sub-databases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While ~77% of Phytophthora EPTs supported the current annotation, a portion of them (7.2% and 12.6% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Energy Technology Data Exchange (ETDEWEB)

Curtis, Bruce A.; Tanifuji, Goro; Burki, Fabien; Gruber, Ansgar; Irimia, Manuuel; Maruyama, Shinichiro; Arias, Maria C.; Ball, Steven G.; Gile, Gillian H.; Hirakawa, Yoshihisa; Hopkins, Julia F.; Kuo, Alan; Rensing, Stefan A.; Schmutz, Jeremy; Symeonidi, Aikaterini; Elias, Marek; Eveleigh, Robert J. M.; Herman, Emily K.; Klute, Mary J.; Nakayama, Takuro; Obornik, Miroslav; Reyes-Prieto, Adrian; Armbrust, E. Virginia; Aves, Stephen J.; Beiko, Robert G.; Coutinho, Pedro; Dacks, Joel B.; Durnford, Dion G.; Fast, Naomi M.; Green, Beverley R.; Grisdale, Cameron J.; Hempel, Franziska; Henrissat, Bernard; Hoppner, Marc P.; Ishida, Ken-Ichiro; Kim, Eunsoo; Koreny, Ludek; Kroth, Peter G.; Liu, Yuan; Malik, Shehre-Banoo; Maier, Uwe G.; McRose, Darcy; Mock, Thomas; Neilson, Jonathan A. D.; Onodera, Naoko T.; Poole, Anthony M.; Pritham, Ellen J.; Richards, Thomas A.; Rocap, Gabrielle; Roy, Scott W.; Sarai, Chihiro; Schaack, Sarah; Shirato, Shu; Slamovits, Claudio H.; Spencer, Davie F.; Suzuki, Shigekatsu; Worden, Alexandra Z.; Zauner, Stefan; Barry, Kerrie; Bell, Callum; Bharti, Arvind K.; Crow, John A.; Grimwood, Jane; Kramer, Robin; Lindquist, Erika; Lucas, Susan; Salamov, Asaf; McFadden, Geoffrey I.; Lane, Christopher E.; Keeling, Patrick J.; Gray, Michael W.; Grigoriev, Igor V.; Archibald, John M.

2012-08-10

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have 21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Pyrosequencing and genetic diversity of microeukaryotes

DEFF Research Database (Denmark)

Harder, Christoffer Bugge

carefully selected waterworks (Article IV), where the bacterial metabolic diversity and its important for water purification was described. Building on this, the most important part of the thesis consists of two pyrosequencing analyses of protozoa with newly developed 18S primers. One specifically targets...... Cercozoa, a particularly abundant phylum of protozoa (Article III), on heath land that had been subjected to prolonged artificially induced drought in a Danish free-air climate-manipulation experiment (CLIMAITE). Article III showed that the testate cercozoan forms responded negatively to prolonged drought...

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

Science.gov (United States)

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

2016-04-07

Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumarchaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Nitrososphaera viennensis and we purified the recombinant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described thermostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches

Energy Technology Data Exchange (ETDEWEB)

Chandonia, John-Marc; Brenner, Steven E.

2004-07-14

The structural genomics project is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy which is medically and biologically relevant, of good value, and tractable. As an option to consider, we present the Pfam5000 strategy, which involves selecting the 5000 most important families from the Pfam database as sources for targets. We compare the Pfam5000 strategy to several other proposed strategies that would require similar numbers of targets. These include including complete solution of several small to moderately sized bacterial proteomes, partial coverage of the human proteome, and random selection of approximately 5000 targets from sequenced genomes. We measure the impact that successful implementation of these strategies would have upon structural interpretation of the proteins in Swiss-Prot, TrEMBL, and 131 complete proteomes (including 10 of eukaryotes) from the Proteome Analysis database at EBI. Solving the structures of proteins from the 5000 largest Pfam families would allow accurate fold assignment for approximately 68 percent of all prokaryotic proteins (covering 59 percent of residues) and 61 percent of eukaryotic proteins (40 percent of residues). More fine-grained coverage which would allow accurate modeling of these proteins would require an order of magnitude more targets. The Pfam5000 strategy may be modified in several ways, for example to focus on larger families, bacterial sequences, or eukaryotic sequences; as long as secondary consideration is given to large families within Pfam, coverage results vary only slightly. In contrast, focusing structural genomics on a single tractable genome would have only a limited impact in structural knowledge of other proteomes: a significant fraction (about 30-40 percent of the proteins, and 40-60 percent of the residues) of each proteome is classified in small

Pyrosequencing of environmental soil samples reveals biodiversity of the Phytophthora resident community in chestnut forests.

Science.gov (United States)

Vannini, Andrea; Bruni, Natalia; Tomassini, Alessia; Franceschini, Selma; Vettraino, Anna Maria

2013-09-01

Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Extensive gene content variation in the Brachypodium distachyon pan-genome correlates with population structure.

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Gordon, Sean P; Contreras-Moreira, Bruno; Woods, Daniel P; Des Marais, David L; Burgess, Diane; Shu, Shengqiang; Stritt, Christoph; Roulin, Anne C; Schackwitz, Wendy; Tyler, Ludmila; Martin, Joel; Lipzen, Anna; Dochy, Niklas; Phillips, Jeremy; Barry, Kerrie; Geuten, Koen; Budak, Hikmet; Juenger, Thomas E; Amasino, Richard; Caicedo, Ana L; Goodstein, David; Davidson, Patrick; Mur, Luis A J; Figueroa, Melania; Freeling, Michael; Catalan, Pilar; Vogel, John P

2017-12-19

While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of differentially present genes in eukaryotes remains poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yield a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g., defense and development), display faster evolutionary rates, lie closer to transposable elements and are less likely to be syntenic with orthologous genes in other grasses. Our data suggest that differentially present genes contribute substantially to phenotypic variation within a eukaryote species, these genes have a major influence in population genetics, and transposable elements play a key role in pan-genome evolution.

Eukaryotic tRNAs fingerprint invertebrates vis-à-vis vertebrates.

Science.gov (United States)

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

2015-01-01

During translation, aminoacyl-tRNA synthetases recognize the identities of the tRNAs to charge them with their respective amino acids. The conserved identities of 58,244 eukaryotic tRNAs of 24 invertebrates and 45 vertebrates in genomic tRNA database were analyzed and their novel features extracted. The internal promoter sequences, namely, A-Box and B-Box, were investigated and evidence gathered that the intervention of optional nucleotides at 17a and 17b correlated with the optimal length of the A-Box. The presence of canonical transcription terminator sequences at the immediate vicinity of tRNA genes was ventured. Even though non-canonical introns had been reported in red alga, green alga, and nucleomorph so far, fairly motivating evidence of their existence emerged in tRNA genes of other eukaryotes. Non-canonical introns were seen to interfere with the internal promoters in two cases, questioning their transcription fidelity. In a first of its kind, phylogenetic constructs based on tRNA molecules delineated and built the trees of the vast and diverse invertebrates and vertebrates. Finally, two tRNA models representing the invertebrates and the vertebrates were drawn, by isolating the dominant consensus in the positional fluctuations of nucleotide compositions.

A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs.

Science.gov (United States)

Swain, Martin T; Tsai, Isheng J; Assefa, Samual A; Newbold, Chris; Berriman, Matthew; Otto, Thomas D

2012-06-07

Genome projects now produce draft assemblies within weeks owing to advanced high-throughput sequencing technologies. For milestone projects such as Escherichia coli or Homo sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects, and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT) that is used to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence and exploit reference genomes (if available) in order to improve scaffolding and generating annotations. The protocol is most accessible for bacterial and small eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes ∼24 h: it doubles the average contig size and annotates over 4,300 gene models.

Mutant power: using mutant allele collections for yeast functional genomics.

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Norman, Kaitlyn L; Kumar, Anuj

2016-03-01

The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Inverse PCR-based method for isolating novel SINEs from genome.

Science.gov (United States)

Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

2014-04-01

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells.

Science.gov (United States)

Daer, René M; Cutts, Josh P; Brafman, David A; Haynes, Karmella A

2017-03-17

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the system's precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

RPAN: rice pan-genome browser for ∼3000 rice genomes.

Science.gov (United States)

Sun, Chen; Hu, Zhiqiang; Zheng, Tianqing; Lu, Kuangchen; Zhao, Yue; Wang, Wensheng; Shi, Jianxin; Wang, Chunchao; Lu, Jinyuan; Zhang, Dabing; Li, Zhikang; Wei, Chaochun

2017-01-25

A pan-genome is the union of the gene sets of all the individuals of a clade or a species and it provides a new dimension of genome complexity with the presence/absence variations (PAVs) of genes among these genomes. With the progress of sequencing technologies, pan-genome study is becoming affordable for eukaryotes with large-sized genomes. The Asian cultivated rice, Oryza sativa L., is one of the major food sources for the world and a model organism in plant biology. Recently, the 3000 Rice Genome Project (3K RGP) sequenced more than 3000 rice genomes with a mean sequencing depth of 14.3×, which provided a tremendous resource for rice research. In this paper, we present a genome browser, Rice Pan-genome Browser (RPAN), as a tool to search and visualize the rice pan-genome derived from 3K RGP. RPAN contains a database of the basic information of 3010 rice accessions, including genomic sequences, gene annotations, PAV information and gene expression data of the rice pan-genome. At least 12 000 novel genes absent in the reference genome were included. RPAN also provides multiple search and visualization functions. RPAN can be a rich resource for rice biology and rice breeding. It is available at http://cgm.sjtu.edu.cn/3kricedb/ or http://www.rmbreeding.cn/pan3k. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases.

Science.gov (United States)

Schwelm, Arne; Fogelqvist, Johan; Knaust, Andrea; Jülke, Sabine; Lilja, Tua; Bonilla-Rosso, German; Karlsson, Magnus; Shevchenko, Andrej; Dhandapani, Vignesh; Choi, Su Ryun; Kim, Hong Gi; Park, Ju Young; Lim, Yong Pyo; Ludwig-Müller, Jutta; Dixelius, Christina

2015-06-18

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

Isolation of 18 Microsatellite Loci in the Desert Mistletoe Phoradendron californicum (Santalaceae Via 454 Pyrosequencing

Directory of Open Access Journals (Sweden)

Juan M. Arroyo

2013-12-01

Full Text Available Premise of the study: Microsatellite primers were developed for the parasitic mistletoe Phoradendron californicum to investigate to what extent population genetic structure depends on host tree distribution within a highly fragmented landscape. Methods and Results: Fourteen unlinked polymorphic and four monomorphic nuclear microsatellite markers were developed using a genomic shotgun pyrosequencing method. A total of 187 alleles plus four monomorphic loci alleles were found in 98 individuals sampled in three populations from the Sonoran Desert in the Baja California peninsula (Mexico. Loci averaged 13.3 alleles per locus (range 4–28, and observed and expected heterozygosities within populations varied from 0.167–0.879 and 0.364–0.932, respectively. Conclusions: Levels of polymorphism of the reported markers are adequate for studies of diversity and fragmentation in natural populations of this parasitic plant. Cross-species amplifications in P. juniperinum and P. diguetianum only showed four markers that could be useful in P. diguetianum.

The impact of genomics on research in diversity and evolution of archaea.

Science.gov (United States)

Mardanov, A V; Ravin, N V

2012-08-01

Since the definition of archaea as a separate domain of life along with bacteria and eukaryotes, they have become one of the most interesting objects of modern microbiology, molecular biology, and biochemistry. Sequencing and analysis of archaeal genomes were especially important for studies on archaea because of a limited availability of genetic tools for the majority of these microorganisms and problems associated with their cultivation. Fifteen years since the publication of the first genome of an archaeon, more than one hundred complete genome sequences of representatives of different phylogenetic groups have been determined. Analysis of these genomes has expanded our knowledge of biology of archaea, their diversity and evolution, and allowed identification and characterization of new deep phylogenetic lineages of archaea. The development of genome technologies has allowed sequencing the genomes of uncultivated archaea directly from enrichment cultures, metagenomic samples, and even from single cells. Insights have been gained into the evolution of key biochemical processes in archaea, such as cell division and DNA replication, the role of horizontal gene transfer in the evolution of archaea, and new relationships between archaea and eukaryotes have been revealed.

Radiotaxons and reliability of a genome

International Nuclear Information System (INIS)

Korogodin, V.I.

1982-01-01

Radiosensitivity of cells (D 0 ) is considered with regard to the structural organization of the genome. The following terms are introduced: ''karyotaxon'', organisms with identical structural organization of the genome, and ''specific genome stability'' K=D 0 C, where C is the quantity of DNA in the cell nucleus; K is the amount of energy (eV) the sorption of which in DNA is necessary and sufficient for one elementary damage to occur. It was shown that Ksub(i)=const. within every karyotaxon ''i''. K 1 =100 eV for viruses, and K 4 =61000 eV for the highest level of genome organization (diploid eukaryotes including man). Potential mechanisms of increasing Ksub(i) with increasing level of genome organization and the role of this factor in evolution are discussed [ru

Classification and Lineage Tracing of SH2 Domains Throughout Eukaryotes.

Science.gov (United States)

Liu, Bernard A

2017-01-01

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, protein domain architecture and the boundary positions between introns and exons within the SH2 domain or genes encoding these domains. Discrete SH2 families can then be traced across various genomes to provide insight into its origins. Furthermore, additional methods for examining potential mechanisms for divergence of SH2 domains from structural changes to alterations in the protein domain content and genome duplication will be discussed. Therefore a better understanding of SH2 domain evolution may enhance our insight into the emergence of phosphotyrosine signaling and the expansion of protein interaction domains.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

Science.gov (United States)

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

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.

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

Science.gov (United States)

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

2011-01-01

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

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

Science.gov (United States)

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-11-20

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

MCM Paradox: Abundance of Eukaryotic Replicative Helicases and Genomic Integrity.

Science.gov (United States)

Das, Mitali; Singh, Sunita; Pradhan, Satyajit; Narayan, Gopeshwar

2014-01-01

As a crucial component of DNA replication licensing system, minichromosome maintenance (MCM) 2-7 complex acts as the eukaryotic DNA replicative helicase. The six related MCM proteins form a heterohexamer and bind with ORC, CDC6, and Cdt1 to form the prereplication complex. Although the MCMs are well known as replicative helicases, their overabundance and distribution patterns on chromatin present a paradox called the "MCM paradox." Several approaches had been taken to solve the MCM paradox and describe the purpose of excess MCMs distributed beyond the replication origins. Alternative functions of these MCMs rather than a helicase had also been proposed. This review focuses on several models and concepts generated to solve the MCM paradox coinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormant origins as a backup during replication stress. Finally, we extend our view towards the effect of alteration of MCM level. Though an excess MCM constituent is needed for normal cells to withstand stress, there must be a delineation of the threshold level in normal and malignant cells. This review also outlooks the future prospects to better understand the MCM biology.

Phylogenetic tree based on complete genomes using fractal and correlation analyses without sequence alignment

Directory of Open Access Journals (Sweden)

Zu-Guo Yu

2006-06-01

Full Text Available The complete genomes of living organisms have provided much information on their phylogenetic relationships. Similarly, the complete genomes of chloroplasts have helped resolve the evolution of this organelle in photosynthetic eukaryotes. In this review, we describe two algorithms to construct phylogenetic trees based on the theories of fractals and dynamic language using complete genomes. These algorithms were developed by our research group in the past few years. Our distance-based phylogenetic tree of 109 prokaryotes and eukaryotes agrees with the biologists' "tree of life" based on the 16S-like rRNA genes in a majority of basic branchings and most lower taxa. Our phylogenetic analysis also shows that the chloroplast genomes are separated into two major clades corresponding to chlorophytes s.l. and rhodophytes s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution.

Hypothesis: Gene-rich plastid genomes in red algae may be an outcome of nuclear genome reduction.

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Qiu, Huan; Lee, Jun Mo; Yoon, Hwan Su; Bhattacharya, Debashish

2017-06-01

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non-photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage. © 2017 Phycological Society of America.

Freedom and Responsibility in Synthetic Genomics: The Synthetic Yeast Project

OpenAIRE

Sliva, Anna; Yang, Huanming; Boeke, Jef D.; Mathews, Debra J. H.

2015-01-01

First introduced in 2011, the Synthetic Yeast Genome (Sc2.0) Project is a large international synthetic genomics project that will culminate in the first eukaryotic cell (Saccharomyces cerevisiae) with a fully synthetic genome. With collaborators from across the globe and from a range of institutions spanning from do-it-yourself biology (DIYbio) to commercial enterprises, it is important that all scientists working on this project are cognizant of the ethical and policy issues associated with...

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha

2015-07-30

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

2015-01-01

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

Science.gov (United States)

Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

2014-01-01

Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

Evolution of endogenous non-retroviral genes integrated into plant genomes

Directory of Open Access Journals (Sweden)

Hyosub Chu

2014-08-01

Full Text Available Numerous comparative genome analyses have revealed the wide extent of horizontal gene transfer (HGT in living organisms, which contributes to their evolution and genetic diversity. Viruses play important roles in HGT. Endogenous viral elements (EVEs are defined as viral DNA sequences present within the genomes of non-viral organisms. In eukaryotic cells, the majority of EVEs are derived from RNA viruses using reverse transcription. In contrast, endogenous non-retroviral elements (ENREs are poorly studied. However, the increasing availability of genomic data and the rapid development of bioinformatics tools have enabled the identification of several ENREs in various eukaryotic organisms. To date, a small number of ENREs integrated into plant genomes have been identified. Of the known non-retroviruses, most identified ENREs are derived from double-strand (ds RNA viruses, followed by single-strand (ss DNA and ssRNA viruses. At least eight virus families have been identified. Of these, viruses in the family Partitiviridae are dominant, followed by viruses of the families Chrysoviridae and Geminiviridae. The identified ENREs have been primarily identified in eudicots, followed by monocots. In this review, we briefly discuss the current view on non-retroviral sequences integrated into plant genomes that are associated with plant-virus evolution and their possible roles in antiviral resistance.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

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Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

2016-01-01

Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

Genomics of Volvocine Algae

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Umen, James G.; Olson, Bradley J.S.C.

2015-01-01

Volvocine algae are a group of chlorophytes that together comprise a unique model for evolutionary and developmental biology. The species Chlamydomonas reinhardtii and Volvox carteri represent extremes in morphological diversity within the Volvocine clade. Chlamydomonas is unicellular and reflects the ancestral state of the group, while Volvox is multicellular and has evolved numerous innovations including germ-soma differentiation, sexual dimorphism, and complex morphogenetic patterning. The Chlamydomonas genome sequence has shed light on several areas of eukaryotic cell biology, metabolism and evolution, while the Volvox genome sequence has enabled a comparison with Chlamydomonas that reveals some of the underlying changes that enabled its transition to multicellularity, but also underscores the subtlety of this transition. Many of the tools and resources are in place to further develop Volvocine algae as a model for evolutionary genomics. PMID:25883411

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

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

2016-01-01

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. PMID:26907343

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.

Allele-Specific DNA Methylation Detection by Pyrosequencing®

DEFF Research Database (Denmark)

Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

2015-01-01

DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

Structural-Functional Organization of the Eukaryotic Cell Nucleus and Transcription Regulation: Introduction to This Special Issue of Biochemistry (Moscow).

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Razin, S V

2018-04-01

This issue of Biochemistry (Moscow) is devoted to the cell nucleus and mechanisms of transcription regulation. Over the years, biochemical processes in the cell nucleus have been studied in isolation, outside the context of their spatial organization. Now it is clear that segregation of functional processes within a compartmentalized cell nucleus is very important for the implementation of basic genetic processes. The functional compartmentalization of the cell nucleus is closely related to the spatial organization of the genome, which in turn plays a key role in the operation of epigenetic mechanisms. In this issue of Biochemistry (Moscow), we present a selection of review articles covering the functional architecture of the eukaryotic cell nucleus, the mechanisms of genome folding, the role of stochastic processes in establishing 3D architecture of the genome, and the impact of genome spatial organization on transcription regulation.

Are maternal mitochondria the selfish entities that are masters of the cells of eukaryotic multicellular organisms?

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Barlow, Peter W; Baldelli, E; Baluška, Frantisek

2009-01-01

The Energide concept, as well as the endosymbiotic theory of eukaryotic cell organization and evolution, proposes that present-day cells of eukaryotic organisms are mosaics of specialized and cooperating units, or organelles. Some of these units were originally free-living prokaryotes, which were engulfed during evolutionary time. Mitochondria represent one of these types of previously independent organisms, the Energide, is another type. This new perspective on the organization of the cell has been further expanded to reveal the concept of a public milieu, the cytosol, in which Energides and mitochondria live, each with their own private internal milieu. The present paper discusses how the endosymbiotic theory implicates a new hypothesis about the hierarchical and communicational organization of the integrated prokaryotic components of the eukaryotic cell and provides a new angle from which to consider the theory of evolution and its bearing upon cellular complexity. Thus, it is proposed that the “selfish gene” hypothesis of Dawkins1 is not the only possible perspective for comprehending genomic and cellular evolution. Our proposal is that maternal mitochondria are the selfish “master” entities of the eukaryotic cell with respect not only to their propagation from cell-to-cell and from generation-to-generation but also to their regulation of all other cellular functions. However, it should be recognized that the concept of “master” and “servant” cell components is a metaphor; in present-day living organisms their organellar components are considered to be interdependent and inseparable. PMID:19513277

Multiple recent horizontal transfers of a large genomic region in cheese making fungi.

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Cheeseman, Kevin; Ropars, Jeanne; Renault, Pierre; Dupont, Joëlle; Gouzy, Jérôme; Branca, Antoine; Abraham, Anne-Laure; Ceppi, Maurizio; Conseiller, Emmanuel; Debuchy, Robert; Malagnac, Fabienne; Goarin, Anne; Silar, Philippe; Lacoste, Sandrine; Sallet, Erika; Bensimon, Aaron; Giraud, Tatiana; Brygoo, Yves

2014-01-01

While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti--called Wallaby--present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes.

Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

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Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

2004-03-01

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Six subgroups and extensive recent duplications characterize the evolution of the eukaryotic tubulin protein family.

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Findeisen, Peggy; Mühlhausen, Stefanie; Dempewolf, Silke; Hertzog, Jonny; Zietlow, Alexander; Carlomagno, Teresa; Kollmar, Martin

2014-08-27

Tubulins belong to the most abundant proteins in eukaryotes providing the backbone for many cellular substructures like the mitotic and meiotic spindles, the intracellular cytoskeletal network, and the axonemes of cilia and flagella. Homologs have even been reported for archaea and bacteria. However, a taxonomically broad and whole-genome-based analysis of the tubulin protein family has never been performed, and thus, the number of subfamilies, their taxonomic distribution, and the exact grouping of the supposed archaeal and bacterial homologs are unknown. Here, we present the analysis of 3,524 tubulins from 504 species. The tubulins formed six major subfamilies, α to ζ. Species of all major kingdoms of the eukaryotes encode members of these subfamilies implying that they must have already been present in the last common eukaryotic ancestor. The proposed archaeal homologs grouped together with the bacterial TubZ proteins as sister clade to the FtsZ proteins indicating that tubulins are unique to eukaryotes. Most species contained α- and/or β-tubulin gene duplicates resulting from recent branch- and species-specific duplication events. This shows that tubulins cannot be used for constructing species phylogenies without resolving their ortholog-paralog relationships. The many gene duplicates and also the independent loss of the δ-, ε-, or ζ-tubulins, which have been shown to be part of the triplet microtubules in basal bodies, suggest that tubulins can functionally substitute each other. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genomes, Phylogeny, and Evolutionary Systems Biology

Energy Technology Data Exchange (ETDEWEB)

Medina, Monica

2005-03-25

With the completion of the human genome and the growing number of diverse genomes being sequenced, a new age of evolutionary research is currently taking shape. The myriad of technological breakthroughs in biology that are leading to the unification of broad scientific fields such as molecular biology, biochemistry, physics, mathematics and computer science are now known as systems biology. Here I present an overview, with an emphasis on eukaryotes, of how the postgenomics era is adopting comparative approaches that go beyond comparisons among model organisms to shape the nascent field of evolutionary systems biology.

Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

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Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

2011-10-01

The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

A Three-Dimensional Model of the Yeast Genome

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Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Complete genome sequence of the myxobacterium Sorangium cellulosum

DEFF Research Database (Denmark)

Schneiker, S; Perlova, O; Kaiser, O

2007-01-01

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum Soce56, which produces several natural products and has...... morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between...... these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism...

Morphological and ecological complexity in early eukaryotic ecosystems.

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Javaux, E J; Knoll, A H; Walter, M R

2001-07-05

Molecular phylogeny and biogeochemistry indicate that eukaryotes differentiated early in Earth history. Sequence comparisons of small-subunit ribosomal RNA genes suggest a deep evolutionary divergence of Eukarya and Archaea; C27-C29 steranes (derived from sterols synthesized by eukaryotes) and strong depletion of 13C (a biogeochemical signature of methanogenic Archaea) in 2,700 Myr old kerogens independently place a minimum age on this split. Steranes, large spheroidal microfossils, and rare macrofossils of possible eukaryotic origin occur in Palaeoproterozoic rocks. Until now, however, evidence for morphological and taxonomic diversification within the domain has generally been restricted to very late Mesoproterozoic and Neoproterozoic successions. Here we show that the cytoskeletal and ecological prerequisites for eukaryotic diversification were already established in eukaryotic microorganisms fossilized nearly 1,500 Myr ago in shales of the early Mesoproterozoic Roper Group in northern Australia.

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

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

2003-01-01

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

Comparing the Dictyostelium and Entamoeba genomes reveals an ancient split in the Conosa lineage.

Directory of Open Access Journals (Sweden)

Jie Song

2005-12-01

Full Text Available The Amoebozoa are a sister clade to the fungi and the animals, but are poorly sampled for completely sequenced genomes. The social amoeba Dictyostelium discoideum and amitochondriate pathogen Entamoeba histolytica are the first Amoebozoa with genomes completely sequenced. Both organisms are classified under the Conosa subphylum. To identify Amoebozoa-specific genomic elements, we compared these two genomes to each other and to other eukaryotic genomes. An expanded phylogenetic tree built from the complete predicted proteomes of 23 eukaryotes places the two amoebae in the same lineage, although the divergence is estimated to be greater than that between animals and fungi, and probably happened shortly after the Amoebozoa split from the opisthokont lineage. Most of the 1,500 orthologous gene families shared between the two amoebae are also shared with plant, animal, and fungal genomes. We found that only 42 gene families are distinct to the amoeba lineage; among these are a large number of proteins that contain repeats of the FNIP domain, and a putative transcription factor essential for proper cell type differentiation in D. discoideum. These Amoebozoa-specific genes may be useful in the design of novel diagnostics and therapies for amoebal pathologies.

Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

KAUST Repository

Prest, Emmanuelle I E C

2014-10-01

The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

Science.gov (United States)

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Pyrosequencing Analysis of Cryogenically Ground Samples from Primary and Secondary/Persistent Endodontic Infections.

Science.gov (United States)

Keskin, Cangül; Demiryürek, Ebru Özsezer; Onuk, Ertan Emek

2017-08-01

This study aimed to characterize the microbial communities of primary and secondary/persistent endodontic infections using high-throughput pyrosequencing from the pulverized samples. The roots of 20 extracted human teeth with primary endodontic infection and 20 teeth with secondary/persistent endodontic infection were collected. The outer surfaces of the roots were disinfected, and whole roots were cryopulverized. 16S amplicon pyrosequencing data from the DNA extracted from the pulverized root powders were obtained, and microorganism abundance and diversity were calculated. Data were analyzed using statistical and bioinformatic methods. Pyrosequencing analysis resulted a total of 2,606,128 sequences from 40 samples. A total of 15 phyla, 160 genera, and 368 species were detected. No significant difference between primary and secondary/persistent endodontic infections was found regarding the diversity and richness of operational taxonomic units at the phyla, genera, and species levels (P > .005). The present study revealed that the microbial diversity of secondary/persistent endodontic infections did not differ than those of primary endodontic infections. A new archaeal species, Candidatus Nitrosoarchaeum limnia, was detected in root canals of 1 patient with primary endodontic infection for the first time. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

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Jayakumar, Vasanthan; Sakakibara, Yasubumi

2017-11-03

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

Directory of Open Access Journals (Sweden)

Tongjit Thanchomnang

Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

The complete mitochondrial genome of the deep-sea sponge Poecillastra laminaris (Astrophorida, Vulcanellidae).

Science.gov (United States)

Zeng, Cong; Thomas, Leighton J; Kelly, Michelle; Gardner, Jonathan P A

2016-05-01

The complete mitochondrial genome of a New Zealand specimen of the deep-sea sponge Poecillastra laminaris (Sollas, 1886) (Astrophorida, Vulcanellidae), from the Colville Ridge, New Zealand, was sequenced using the 454 Life Science pyrosequencing system. To identify homologous mitochondrial sequences, the 454 reads were mapped to the complete mitochondrial genome sequence of Geodia neptuni (GeneBank No. NC_006990). The P. laminaris genome is 18,413 bp in length and includes 14 protein-coding genes, 24 transfer RNA genes and 2 ribosomal RNA genes. Gene order resembled that of other demosponges. The base composition of the genome is A (29.1%), T (35.2%), C (14.0%) and G (21.7%). This is the second published mitogenome for a sponge of the order Astrophorida and will be useful in future phylogenetic analysis of deep-sea sponges.

[Structure and evolution of the eukaryotic FANCJ-like proteins].

Science.gov (United States)

Wuhe, Jike; Zefeng, Wu; Sanhong, Fan; Xuguang, Xi

2015-02-01

The FANCJ-like protein family is a class of ATP-dependent helicases that can catalytically unwind duplex DNA along the 5'-3' direction. It is involved in the processes of DNA damage repair, homologous recombination and G-quadruplex DNA unwinding, and plays a critical role in maintaining genome integrity. In this study, we systemically analyzed FNACJ-like proteins from 47 eukaryotic species and discussed their sequences diversity, origin and evolution, motif organization patterns and spatial structure differences. Four members of FNACJ-like proteins, including XPD, CHL1, RTEL1 and FANCJ, were found in eukaryotes, but some of them were seriously deficient in most fungi and some insects. For example, the Zygomycota fungi lost RTEL1, Basidiomycota and Ascomycota fungi lost RTEL1 and FANCJ, and Diptera insect lost FANCJ. FANCJ-like proteins contain canonical motor domains HD1 and HD2, and the HD1 domain further integrates with three unique domains Fe-S, Arch and Extra-D. Fe-S and Arch domains are relatively conservative in all members of the family, but the Extra-D domain is lost in XPD and differs from one another in rest members. There are 7, 10 and 2 specific motifs found from the three unique domains respectively, while 5 and 12 specific motifs are found from HD1 and HD2 domains except the conserved motifs reported previously. By analyzing the arrangement pattern of these specific motifs, we found that RTEL1 and FANCJ are more closer and share two specific motifs Vb2 and Vc in HD2 domain, which are likely related with their G-quadruplex DNA unwinding activity. The evidence of evolution showed that FACNJ-like proteins were originated from a helicase, which has a HD1 domain inserted by extra Fe-S domain and Arch domain. By three continuous gene duplication events and followed specialization, eukaryotes finally possessed the current four members of FANCJ-like proteins.

Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

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David S. Shin

2014-01-01

Full Text Available As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.

Eukaryotes first: how could that be?

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Mariscal, Carlos; Doolittle, W Ford

2015-09-26

In the half century since the formulation of the prokaryote : eukaryote dichotomy, many authors have proposed that the former evolved from something resembling the latter, in defiance of common (and possibly common sense) views. In such 'eukaryotes first' (EF) scenarios, the last universal common ancestor is imagined to have possessed significantly many of the complex characteristics of contemporary eukaryotes, as relics of an earlier 'progenotic' period or RNA world. Bacteria and Archaea thus must have lost these complex features secondarily, through 'streamlining'. If the canonical three-domain tree in which Archaea and Eukarya are sisters is accepted, EF entails that Bacteria and Archaea are convergently prokaryotic. We ask what this means and how it might be tested. © 2015 The Author(s).

Whole-genome pyrosequencing of an epidemic multidrug-resistant Acinetobacter baumannii strain belonging to the European clone II group

DEFF Research Database (Denmark)

Iacono, M.; Villa, L.; Fortini, D.

2008-01-01

The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated bla(OXA-58) carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes...

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

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Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia

2013-01-01

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336

Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

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Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

2014-10-15

The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data

DEFF Research Database (Denmark)

Rask, Thomas Salhøj; Petersen, Bent; Chen, Donald S.

2016-01-01

. The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data....... This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene...... family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon...

The origin of the eukaryotic cell

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Hartman, H.

1984-01-01

The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.

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

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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. © 2015 John Wiley & Sons Ltd.

A sensitive issue: Pyrosequencing as a valuable forensic SNP typing platform

DEFF Research Database (Denmark)

Harrison, C.; Musgrave-Brown, E.; Bender, K.

2006-01-01

Analysing minute amounts of DNA is a routine challenge in forensics in part due to the poor sensitivity of an instrument and its inability to detect results from forensic samples. In this study, the sensitivity of the Pyrosequencing method is investigated using varying concentrations of DNA and f...

Initial genomics of the human nucleolus.

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Attila Németh

2010-03-01

Full Text Available We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD-localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD-specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture.

Initial Genomics of the Human Nucleolus

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Németh, Attila; Conesa, Ana; Santoyo-Lopez, Javier; Medina, Ignacio; Montaner, David; Péterfia, Bálint; Solovei, Irina; Cremer, Thomas; Dopazo, Joaquin; Längst, Gernot

2010-01-01

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture. PMID:20361057

Polyploidy: adaptation to the genomic environment.

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Hollister, Jesse D

2015-02-01

Genomic evidence of ancestral whole genome duplication (WGD) and polyploidy is widespread among eukaryotic species, and especially among plants. WGD is thought to provide the raw material for adaptation in the form of duplicated genes, and polyploids are thought to benefit from both physiological and genetic buffering. Comparatively little attention has focused on the genomic challenge of polyploidy, however, although much evidence exists that polyploidy severely perturbs important cellular functions. Here, I review recent progress in the study of the re-establishment of stable meiosis in recently evolved polyploids, focusing on four plant species. This work has yielded an insight into the mechanisms underlying stabilization of genome transmission in polyploids, and is revealing remarkable parallels among diverse taxa. Importantly, these studies also provide a road map for investigating how polyploids respond to the challenge of WGD.

Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing

DEFF Research Database (Denmark)

Masoud, Wafa Mahmoud Hasan; Takamiya, Monica K Wik; Vogensen, Finn Kvist

2011-01-01

ripening. Other bacteria like Corynebacterium, Halomonas, Pediococcus, Micrococcus and Staphylococcus, which were encountered in some cheese samples at low percentages compared with the total bacterial populations, were only detected by pyrosequencing. 16S rRNA gene pyrosequencing is an efficient method...

Organizational heterogeneity of vertebrate genomes.

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Frenkel, Svetlana; Kirzhner, Valery; Korol, Abraham

2012-01-01

Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS) analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers) in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM) allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.

Organizational heterogeneity of vertebrate genomes.

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Svetlana Frenkel

Full Text Available Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.

2012 U.S. Department of Energy: Joint Genome Institute: Progress Report

Energy Technology Data Exchange (ETDEWEB)

Gilbert, David [DOE JGI Public Affairs Manager

2013-01-01

The mission of the U.S. Department of Energy Joint Genome Institute (DOE JGI) is to serve the diverse scientific community as a user facility, enabling the application of large-scale genomics and analysis of plants, microbes, and communities of microbes to address the DOE mission goals in bioenergy and the environment. The DOE JGI's sequencing efforts fall under the Eukaryote Super Program, which includes the Plant and Fungal Genomics Programs; and the Prokaryote Super Program, which includes the Microbial Genomics and Metagenomics Programs. In 2012, several projects made news for their contributions to energy and environment research.

Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae.

Science.gov (United States)

Wijffels, René H; Kruse, Olaf; Hellingwerf, Klaas J

2013-06-01

Both cyanobacteria and eukaryotic microalgae are promising organisms for sustainable production of bulk products such as food, feed, materials, chemicals and fuels. In this review we will summarize the potential and current biotechnological developments. Cyanobacteria are promising host organisms for the production of small molecules that can be secreted such as ethanol, butanol, fatty acids and other organic acids. Eukaryotic microalgae are interesting for products for which cellular storage is important such as proteins, lipids, starch and alkanes. For the development of new and promising lines of production, strains of both cyanobacteria and eukaryotic microalgae have to be improved. Transformation systems have been much better developed in cyanobacteria. However, several products would be preferably produced with eukaryotic microalgae. In the case of cyanobacteria a synthetic-systems biology approach has a great potential to exploit cyanobacteria as cell factories. For eukaryotic microalgae transformation systems need to be further developed. A promising strategy is transformation of heterologous (prokaryotic and eukaryotic) genes in established eukaryotic hosts such as Chlamydomonas reinhardtii. Experimental outdoor pilots under containment for the production of genetically modified cyanobacteria and microalgae are in progress. For full scale production risks of release of genetically modified organisms need to be assessed. Copyright © 2013. Published by Elsevier Ltd.

Death of a dogma: eukaryotic mRNAs can code for more than one protein.

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Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

2016-01-08

mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

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Binhua Liang

Full Text Available BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey

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Varala Kranthi

2007-05-01

Full Text Available Abstract Background Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA. Results We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis. Conclusion This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.

xGDBvm: A Web GUI-Driven Workflow for Annotating Eukaryotic Genomes in the Cloud.

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Duvick, Jon; Standage, Daniel S; Merchant, Nirav; Brendel, Volker P

2016-04-01

Genome-wide annotation of gene structure requires the integration of numerous computational steps. Currently, annotation is arguably best accomplished through collaboration of bioinformatics and domain experts, with broad community involvement. However, such a collaborative approach is not scalable at today's pace of sequence generation. To address this problem, we developed the xGDBvm software, which uses an intuitive graphical user interface to access a number of common genome analysis and gene structure tools, preconfigured in a self-contained virtual machine image. Once their virtual machine instance is deployed through iPlant's Atmosphere cloud services, users access the xGDBvm workflow via a unified Web interface to manage inputs, set program parameters, configure links to high-performance computing (HPC) resources, view and manage output, apply analysis and editing tools, or access contextual help. The xGDBvm workflow will mask the genome, compute spliced alignments from transcript and/or protein inputs (locally or on a remote HPC cluster), predict gene structures and gene structure quality, and display output in a public or private genome browser complete with accessory tools. Problematic gene predictions are flagged and can be reannotated using the integrated yrGATE annotation tool. xGDBvm can also be configured to append or replace existing data or load precomputed data. Multiple genomes can be annotated and displayed, and outputs can be archived for sharing or backup. xGDBvm can be adapted to a variety of use cases including de novo genome annotation, reannotation, comparison of different annotations, and training or teaching. © 2016 American Society of Plant Biologists. All rights reserved.

Genome Sequence of the Biocontrol Strain Pseudomonas fluorescens F113

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Redondo-Nieto, Miguel; Barret, Matthieu; Morrisey, John P.; Germaine, Kieran; Martínez-Granero, Francisco; Barahona, Emma; Navazo, Ana; Sánchez-Contreras, María; Moynihan, Jennifer A.; Giddens, Stephen R.; Coppoolse, Eric R.; Muriel, Candela; Stiekema, Willem J.; Rainey, Paul B.; Dowling, David; O'Gara, Fergal; Martín, Marta

2012-01-01

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms. PMID:22328765

Comparative genomics of phylogenetically diverse unicellular eukaryotes provide new insights into the genetic basis for the evolution of the programmed cell death machinery.

Science.gov (United States)

Nedelcu, Aurora M

2009-03-01

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes--from ligands and receptors to executors of PCD--was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae--used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors.

Identification and characterization of insect-specific proteins by genome data analysis

DEFF Research Database (Denmark)

Zhang, Guojie; Wang, Hongsheng; Shi, Junjie

2007-01-01

melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum, and Apis mellifera were compared to the complete genomes of three non-insect eukaryotes (opisthokonts) Homo sapiens, Caenorhabditis elegans and Saccharomyces cerevisiae. This operation yielded 154 groups of orthologous proteins in Drosophila...

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

NARCIS (Netherlands)

Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Kranenburg, Van Richard; Oost, Van Der John

2017-01-01

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

DEFF Research Database (Denmark)

Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke Fenna

2017-01-01

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including...

Winter-summer succession of unicellular eukaryotes in a meso-eutrophic coastal system.

Science.gov (United States)

Christaki, Urania; Kormas, Konstantinos A; Genitsaris, Savvas; Georges, Clément; Sime-Ngando, Télesphore; Viscogliosi, Eric; Monchy, Sébastien

2014-01-01

The objective of this study was to explore the succession of planktonic unicellular eukaryotes by means of 18S rRNA gene tag pyrosequencing in the eastern English Channel (EEC) during the winter to summer transition. The 59 most representative (>0.1%, representing altogether 95% of total reads), unique operational taxonomic units (OTUs) from all samples belonged to 18 known high-level taxonomic groups and 1 unaffiliated clade. The five most abundant OTUs (69.2% of total reads) belonged to Dinophyceae, Cercozoa, Haptophyceae, marine alveolate group I, and Fungi. Cluster and network analysis between samples distinguished the winter, the pre-bloom, the Phaeocystis globosa bloom and the post-bloom early summer conditions. The OTUs-based network revealed that P. globosa showed a relatively low number of connections-most of them negative-with all other OTUs. Fungi were linked to all major taxonomic groups, except Dinophyceae. Cercozoa mostly co-occurred with the Fungi, the Bacillariophyceae and several of the miscellaneous OTUs. This study provided a more detailed exploration into the planktonic succession pattern of the EEC due to its increased depth of taxonomic sampling over previous efforts based on classical monitoring observations. Data analysis implied that the food web concept in a coastal system based on predator-prey (e.g. grazer-phytoplankton) relationships is just a part of the ecological picture; and those organisms exploiting a variety of strategies, such as saprotrophy and parasitism, are persistent and abundant members of the community.

Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

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Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

2013-08-01

The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Conservation and Variability of Meiosis Across the Eukaryotes.

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Loidl, Josef

2016-11-23

Comparisons among a variety of eukaryotes have revealed considerable variability in the structures and processes involved in their meiosis. Nevertheless, conventional forms of meiosis occur in all major groups of eukaryotes, including early-branching protists. This finding confirms that meiosis originated in the common ancestor of all eukaryotes and suggests that primordial meiosis may have had many characteristics in common with conventional extant meiosis. However, it is possible that the synaptonemal complex and the delicate crossover control related to its presence were later acquisitions. Later still, modifications to meiotic processes occurred within different groups of eukaryotes. Better knowledge on the spectrum of derived and uncommon forms of meiosis will improve our understanding of many still mysterious aspects of the meiotic process and help to explain the evolutionary basis of functional adaptations to the meiotic program.

Wrinkles in the rare biosphere: Pyrosequencing errors can lead to artificial inflation of diversity estimates

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Kunin, Victor; Engelbrektson, Anna; Ochman, Howard; Hugenholtz, Philip

2009-08-01

Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the 'rare biosphere', is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere.

Universal internucleotide statistics in full genomes: a footprint of the DNA structure and packaging?

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Mikhail I Bogachev

Full Text Available Uncovering the fundamental laws that govern the complex DNA structural organization remains challenging and is largely based upon reconstructions from the primary nucleotide sequences. Here we investigate the distributions of the internucleotide intervals and their persistence properties in complete genomes of various organisms from Archaea and Bacteria to H. Sapiens aiming to reveal the manifestation of the universal DNA architecture. We find that in all considered organisms the internucleotide interval distributions exhibit the same [Formula: see text]-exponential form. While in prokaryotes a single [Formula: see text]-exponential function makes the best fit, in eukaryotes the PDF contains additionally a second [Formula: see text]-exponential, which in the human genome makes a perfect approximation over nearly 10 decades. We suggest that this functional form is a footprint of the heterogeneous DNA structure, where the first [Formula: see text]-exponential reflects the universal helical pitch that appears both in pro- and eukaryotic DNA, while the second [Formula: see text]-exponential is a specific marker of the large-scale eukaryotic DNA organization.

Eukaryotic Cell Panorama

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Goodsell, David S.

2011-01-01

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

Interaction of tRNA with Eukaryotic Ribosome

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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.

Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics

DEFF Research Database (Denmark)

Mikkelsen, Martin; Frank-Hansen, Rune; Hansen, Anders Johannes

2014-01-01

RESULTS: of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated...

Identification and characterization of insect-specific proteins by genome data analysis

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Clark Terry

2007-04-01

Full Text Available Abstract Background Insects constitute the vast majority of known species with their importance including biodiversity, agricultural, and human health concerns. It is likely that the successful adaptation of the Insecta clade depends on specific components in its proteome that give rise to specialized features. However, proteome determination is an intensive undertaking. Here we present results from a computational method that uses genome analysis to characterize insect and eukaryote proteomes as an approximation complementary to experimental approaches. Results Homologs in common to Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum, and Apis mellifera were compared to the complete genomes of three non-insect eukaryotes (opisthokonts Homo sapiens, Caenorhabditis elegans and Saccharomyces cerevisiae. This operation yielded 154 groups of orthologous proteins in Drosophila to be insect-specific homologs; 466 groups were determined to be common to eukaryotes (represented by three opisthokonts. ESTs from the hemimetabolous insect Locust migratoria were also considered in order to approximate their corresponding genes in the insect-specific homologs. Stress and stimulus response proteins were found to constitute a higher fraction in the insect-specific homologs than in the homologs common to eukaryotes. Conclusion The significant representation of stress response and stimulus response proteins in proteins determined to be insect-specific, along with specific cuticle and pheromone/odorant binding proteins, suggest that communication and adaptation to environments may distinguish insect evolution relative to other eukaryotes. The tendency for low Ka/Ks ratios in the insect-specific protein set suggests purifying selection pressure. The generally larger number of paralogs in the insect-specific proteins may indicate adaptation to environment changes. Instances in our insect-specific protein set have been arrived at through

Genome Defense Mechanisms in Neurospora and Associated Specialized Proteins

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Ranjan Tamuli

2010-06-01

Full Text Available Neurospora crassa, the filamentous fungus possesses widest array of genome defense mechanisms known to any eukaryotic organism, including a process called repeat-induced point mutation (RIP. RIP is a genome defense mechanism that hypermutates repetitive DNA sequences; analogous to genomic imprinting in mammals. As an impact of RIP, Neurospora possesses many fewer genes in multigene families than expected. A DNA methyltransferase homologue, RID was shown to be essential for RIP. Recently, a variant catalytic subunit of translesion DNA polymerase zeta (Pol zeta has been found to be essential for dominant RIP suppressor phenotype. Meiotic silencing and quelling are two other genome defense mechanisms in Neurospora, and proteins required for these two processes have been identified through genetic screens.

Whole genome duplication affects evolvability of flowering time in an autotetraploid plant.

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Sara L Martin

Full Text Available Whole genome duplications have occurred recurrently throughout the evolutionary history of eukaryotes. The resulting genetic and phenotypic changes can influence physiological and ecological responses to the environment; however, the impact of genome copy number on evolvability has rarely been examined experimentally. Here, we evaluate the effect of genome duplication on the ability to respond to selection for early flowering time in lines drawn from naturally occurring diploid and autotetraploid populations of the plant Chamerion angustifolium (fireweed. We contrast this with the result of four generations of selection on synthesized neoautotetraploids, whose genic variability is similar to diploids but genome copy number is similar to autotetraploids. In addition, we examine correlated responses to selection in all three groups. Diploid and both extant tetraploid and neoautotetraploid lines responded to selection with significant reductions in time to flowering. Evolvability, measured as realized heritability, was significantly lower in extant tetraploids (^b(T =  0.31 than diploids (^b(T =  0.40. Neotetraploids exhibited the highest evolutionary response (^b(T  =  0.55. The rapid shift in flowering time in neotetraploids was associated with an increase in phenotypic variability across generations, but not with change in genome size or phenotypic correlations among traits. Our results suggest that whole genome duplications, without hybridization, may initially alter evolutionary rate, and that the dynamic nature of neoautopolyploids may contribute to the prevalence of polyploidy throughout eukaryotes.

Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

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Antonio Postigo

2017-05-01

Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

An Evolutionary Framework for Understanding the Origin of Eukaryotes

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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.

[MiRNA system in unicellular eukaryotes and its evolutionary implications].

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Zhang, Yan-Qiong; Wen, Jian-Fan

2010-02-01

microRNAs (miRNAs) in higher multicellular eukaryotes have been extensively studied in recent years. Great progresses have also been achieved for miRNAs in unicellular eukaryotes. All these studies not only enrich our knowledge about the complex expression regulation system in diverse organisms, but also have evolutionary significance for understanding the origin of this system. In this review, Authors summarize the recent advance in the studies of miRNA in unicellular eukaryotes, including that on the most primitive unicellular eukaryote--Giardia. The origin and evolution of miRNA system is also discussed.

Complete genome sequence of the fire blight pathogen Erwinia pyrifoliae DSM 12163T and comparative genomic insights into plant pathogenicity

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Frey Jürg E

2010-01-01

Full Text Available Abstract Background Erwinia pyrifoliae is a newly described necrotrophic pathogen, which causes fire blight on Asian (Nashi pear and is geographically restricted to Eastern Asia. Relatively little is known about its genetics compared to the closely related main fire blight pathogen E. amylovora. Results The genome of the type strain of E. pyrifoliae strain DSM 12163T, was sequenced using both 454 and Solexa pyrosequencing and annotated. The genome contains a circular chromosome of 4.026 Mb and four small plasmids. Based on their respective role in virulence in E. amylovora or related organisms, we identified several putative virulence factors, including type III and type VI secretion systems and their effectors, flagellar genes, sorbitol metabolism, iron uptake determinants, and quorum-sensing components. A deletion in the rpoS gene covering the most conserved region of the protein was identified which may contribute to the difference in virulence/host-range compared to E. amylovora. Comparative genomics with the pome fruit epiphyte Erwinia tasmaniensis Et1/99 showed that both species are overall highly similar, although specific differences were identified, for example the presence of some phage gene-containing regions and a high number of putative genomic islands containing transposases in the E. pyrifoliae DSM 12163T genome. Conclusions The E. pyrifoliae genome is an important addition to the published genome of E. tasmaniensis and the unfinished genome of E. amylovora providing a foundation for re-sequencing additional strains that may shed light on the evolution of the host-range and virulence/pathogenicity of this important group of plant-associated bacteria.

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

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Penny David

2007-10-01

Full Text Available Abstract Background 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, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. Results For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. Conclusion Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

Microbial taxonomy in the post-genomic era: Rebuilding from scratch?

Energy Technology Data Exchange (ETDEWEB)

Thompson, Cristiane C. [Univ. of Rio de Janeiro (UFRJ) (Brazil); Amaral, Gilda R. [Univ. of Rio de Janeiro (UFRJ) (Brazil); Campeão, Mariana [Univ. of Rio de Janeiro (UFRJ) (Brazil); Edwards, Robert A. [Univ. of Rio de Janeiro (UFRJ) (Brazil); San Diego State Univ., CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Polz, Martin F. [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Dutilh, Bas E. [Univ. of Rio de Janeiro (UFRJ) (Brazil); Radbould Univ., Nijmegen (Netherlands); Ussery, David W. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Sawabe, Tomoo [Hokkaido Univ., Hakodate (Japan); Swings, Jean [Univ. of Rio de Janeiro (UFRJ) (Brazil); Ghent Univ. (Belgium); Thompson, Fabiano L. [Univ. of Rio de Janeiro (UFRJ) (Brazil); Advanced Systems Laboratory Production Management COPPE / UFRJ, Rio de Janeiro (Brazil)

2014-12-23

Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. We re-evaluated the prokaryote species twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. We will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

Diverse circovirus-like genome architectures revealed by environmental metagenomics.

Science.gov (United States)

Rosario, Karyna; Duffy, Siobain; Breitbart, Mya

2009-10-01

Single-stranded DNA (ssDNA) viruses with circular genomes are the smallest viruses known to infect eukaryotes. The present study identified 10 novel genomes similar to ssDNA circoviruses through data-mining of public viral metagenomes. The metagenomic libraries included samples from reclaimed water and three different marine environments (Chesapeake Bay, British Columbia coastal waters and Sargasso Sea). All the genomes have similarities to the replication (Rep) protein of circoviruses; however, only half have genomic features consistent with known circoviruses. Some of the genomes exhibit a mixture of genomic features associated with different families of ssDNA viruses (i.e. circoviruses, geminiviruses and parvoviruses). Unique genome architectures and phylogenetic analysis of the Rep protein suggest that these viruses belong to novel genera and/or families. Investigating the complex community of ssDNA viruses in the environment can lead to the discovery of divergent species and help elucidate evolutionary links between ssDNA viruses.

Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

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Yongming Wang

2014-03-01

Full Text Available Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB and piggyBac (PB that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1, a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

Eukaryotic cell flattening

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Bae, Albert; Westendorf, Christian; Erlenkamper, Christoph; Galland, Edouard; Franck, Carl; Bodenschatz, Eberhard; Beta, Carsten

2010-03-01

Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field to total internal reflection fluorescence microscopy. In this talk, we will discuss traditional overlay techniques, and more modern, microfluidic based flattening, which provides a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method.

The genome sequence of the model ascomycete fungus Podospora anserina

NARCIS (Netherlands)

Espagne, Eric; Lespinet, Olivier; Malagnac, Fabienne; Da Silva, Corinne; Jaillon, Olivier; Porcel, Betina M; Couloux, Arnaud; Aury, Jean-Marc; Ségurens, Béatrice; Poulain, Julie; Anthouard, Véronique; Grossetete, Sandrine; Khalili, Hamid; Coppin, Evelyne; Déquard-Chablat, Michelle; Picard, Marguerite; Contamine, Véronique; Arnaise, Sylvie; Bourdais, Anne; Berteaux-Lecellier, Véronique; Gautheret, Daniel; de Vries, Ronald P; Battaglia, Evy; Coutinho, Pedro M; Danchin, Etienne Gj; Henrissat, Bernard; Khoury, Riyad El; Sainsard-Chanet, Annie; Boivin, Antoine; Pinan-Lucarré, Bérangère; Sellem, Carole H; Debuchy, Robert; Wincker, Patrick; Weissenbach, Jean; Silar, Philippe

2008-01-01

BACKGROUND: The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. RESULTS: We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed

Genome-wide study of correlations between genomic features and their relationship with the regulation of gene expression.

Science.gov (United States)

Kravatsky, Yuri V; Chechetkin, Vladimir R; Tchurikov, Nikolai A; Kravatskaya, Galina I

2015-02-01

The broad class of tasks in genetics and epigenetics can be reduced to the study of various features that are distributed over the genome (genome tracks). The rapid and efficient processing of the huge amount of data stored in the genome-scale databases cannot be achieved without the software packages based on the analytical criteria. However, strong inhomogeneity of genome tracks hampers the development of relevant statistics. We developed the criteria for the assessment of genome track inhomogeneity and correlations between two genome tracks. We also developed a software package, Genome Track Analyzer, based on this theory. The theory and software were tested on simulated data and were applied to the study of correlations between CpG islands and transcription start sites in the Homo sapiens genome, between profiles of protein-binding sites in chromosomes of Drosophila melanogaster, and between DNA double-strand breaks and histone marks in the H. sapiens genome. Significant correlations between transcription start sites on the forward and the reverse strands were observed in genomes of D. melanogaster, Caenorhabditis elegans, Mus musculus, H. sapiens, and Danio rerio. The observed correlations may be related to the regulation of gene expression in eukaryotes. Genome Track Analyzer is freely available at http://ancorr.eimb.ru/. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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

Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

Science.gov (United States)

Zellweger, Ralph; Lopes, Massimo

2018-01-01

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

Science.gov (United States)

Song, Qinxin; Wei, Guijiang; Zhou, Guohua

2014-07-01

A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

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Yann Marcy

2007-09-01

Full Text Available Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

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......, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations....... 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...

Potential human pathogenic bacteria in a mixed urban watershed as revealed by pyrosequencing.

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A Mark Ibekwe

Full Text Available Current microbial source tracking (MST methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs, recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP, Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%, agricultural runoff sediment (6.52%, and Prado Park sediment (6.00%, respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78-4.08%. Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.

Insertion Sequence-Caused Large Scale-Rearrangements in the Genome of Escherichia coli

Science.gov (United States)

2016-07-18

affordable ap- proach to genome-wide characterization of genetic varia - tion in bacterial and eukaryotic genomes (1–3). In addition to small-scale...Paired-End Reads), that uses a graph-based al- gorithm (27) capable of detecting most large-scale varia - tion involving repetitive regions, including novel...Avila,P., Grinsted,J. and De La Cruz,F. (1988) Analysis of the variable endpoints generated by one-ended transposition of Tn21.. J. Bacteriol., 170

Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes communities in the gut microbiota of obese humans

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Raoult Didier

2008-12-01

Full Text Available Abstract Background Recent studies of 16S rRNA genes in the mammalian gut microbiota distinguished a higher Firmicutes/Bacteroidetes ratio in obese individuals compared to lean individuals. This ratio was estimated using a clonal Sanger sequencing approach which is time-consuming and requires laborious data analysis. In contrast, new high-throughput pyrosequencing technology offers an inexpensive alternative to clonal Sanger sequencing and would significantly advance our understanding of obesity via the development of a clinical diagnostic method. Here we present a cost-effective method that combines 16S rRNA pyrosequencing and DNA barcodes of the Firmicutes and Bacteroidetes 16S rRNA genes to determine the Firmicutes/Bacteroidetes ratio in the gut microbiota of obese humans. Results The main result was the identification of DNA barcodes targeting the Firmicutes and Bacteroidetes phyla. These barcodes were validated using previously published 16S rRNA gut microbiota clone libraries. In addition, an accurate F/B ratio was found when the DNA barcodes were applied to short pyrosequencing reads of published gut metagenomes. Finally, the barcodes were utilized to define the F/B ratio of 16S rRNA pyrosequencing data generated from brain abscess pus and cystic fibrosis sputum. Conclusion Using DNA barcodes of Bacteroidetes and Firmicutes 16S rRNA genes combined with pyrosequencing is a cost-effective method for monitoring relevant changes in the relative abundance of Firmicutes and Bacteroidetes bacterial communities in microbial ecosystems.

xGDBvm: A Web GUI-Driven Workflow for Annotating Eukaryotic Genomes in the Cloud[OPEN

Science.gov (United States)

Merchant, Nirav

2016-01-01

Genome-wide annotation of gene structure requires the integration of numerous computational steps. Currently, annotation is arguably best accomplished through collaboration of bioinformatics and domain experts, with broad community involvement. However, such a collaborative approach is not scalable at today’s pace of sequence generation. To address this problem, we developed the xGDBvm software, which uses an intuitive graphical user interface to access a number of common genome analysis and gene structure tools, preconfigured in a self-contained virtual machine image. Once their virtual machine instance is deployed through iPlant’s Atmosphere cloud services, users access the xGDBvm workflow via a unified Web interface to manage inputs, set program parameters, configure links to high-performance computing (HPC) resources, view and manage output, apply analysis and editing tools, or access contextual help. The xGDBvm workflow will mask the genome, compute spliced alignments from transcript and/or protein inputs (locally or on a remote HPC cluster), predict gene structures and gene structure quality, and display output in a public or private genome browser complete with accessory tools. Problematic gene predictions are flagged and can be reannotated using the integrated yrGATE annotation tool. xGDBvm can also be configured to append or replace existing data or load precomputed data. Multiple genomes can be annotated and displayed, and outputs can be archived for sharing or backup. xGDBvm can be adapted to a variety of use cases including de novo genome annotation, reannotation, comparison of different annotations, and training or teaching. PMID:27020957

Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

Science.gov (United States)

2010-08-25

in honey bee colony collapse disorder. Science 318: 283–287. 39. Towner JS, Sealy TK, Khristova ML, Albarino CG, Conlan S, et al. (2008) Newly...utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations...detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

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Kamila Maliszewska-Olejniczak

2015-07-01

Full Text Available Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs. Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium

Genome size of 14 species of fireflies (Insecta, Coleoptera, Lampyridae

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Gui-Chun Liu

2017-11-01

Full Text Available Eukaryotic genome size data are important both as the basis for comparative research into genome evolution and as estimators of the cost and difficulty of genome sequencing programs for non-model organisms. In this study, the genome size of 14 species of fireflies (Lampyridae (two genera in Lampyrinae, three genera in Luciolinae, and one genus in subfamily incertae sedis were estimated by propidium iodide (PI-based flow cytometry. The haploid genome sizes of Lampyridae ranged from 0.42 to 1.31 pg, a 3.1-fold span. Genome sizes of the fireflies varied within the tested subfamilies and genera. Lamprigera and Pyrocoelia species had large and small genome sizes, respectively. No correlation was found between genome size and morphological traits such as body length, body width, eye width, and antennal length. Our data provide additional information on genome size estimation of the firefly family Lampyridae. Furthermore, this study will help clarify the cost and difficulty of genome sequencing programs for non-model organisms and will help promote studies on firefly genome evolution.

Synthesis of eukaryotic lipid biomarkers in the bacterial domain

Science.gov (United States)

Welander, P. V.; Banta, A. B.; Lee, A. K.; Wei, J. H.

2017-12-01

Lipid biomarkers are organic molecules preserved in sediments and sedimentary rocks that can function as geological proxies for certain microbial taxa or for specific environmental conditions. These molecular fossils provide a link between organisms and their environments in both modern and ancient settings and have afforded significant insight into ancient climatic events, mass extinctions, and various evolutionary transitions throughout Earth's history. However, the proper interpretation of lipid biomarkers is dependent on a broad understanding of their diagenetic precursors in modern systems. This includes understanding the taphonomic transformations that these molecules undergo, their biosynthetic pathways, and the ecological conditions that affect their cellular production. In this study, we focus on one group of lipid biomarkers - the sterols. These are polycyclic isoprenoidal lipids that have a high preservation potential and play a critical role in the physiology of most eukaryotes. However, the synthesis and function of these lipids in the bacterial domain has not been fully explored. Here we utilize a combination of bioinformatics, microbial genetics, and biochemistry to demonstrate that bacterial sterol producers are more prevalent in environmental metagenomic samples than in the genomic databases of cultured organisms and to identify novel proteins required to synthesize and modify sterols in bacteria. These proteins represent a distinct pathway for sterol synthesis exclusive to bacteria and indicate that sterol synthesis in bacteria may have evolved independently of eukaryotic sterol biosynthesis. Taken together, these results demonstrate how studies in extant bacteria can provide insight into the biological sources and the biosynthetic pathways of specific lipid biomarkers and in turn may allow for more robust interpretation of biomarker signatures.

AUG is the only initiation codon in eukaryotes

Energy Technology Data Exchange (ETDEWEB)

Sherman, F; McKnight, G; Stewart, J W

1980-01-01

An analysis of mutants of the yeast Saccharomyces cerevisiae indicates that AUG is the sole codon capable of initiating translation of iso-1-cytochrome c. This result with yeast and the sequence results of numerous eukaryotic genes indicate that AUG is the only initiation codon in eukaryotes; in contrast, results with Escherichia colia and bacteriophages indicate that both AUG and GUG are initiation codons in prokaryotes. The difference can be explained by the lack of the t/sup 6/ A hypermodified nucleoside (N-(9-(..beta..-D-ribofuranosyl)purin-6-ylcarbamoyl)threonine) in prokaryotic initiator tRNA and its presence in eukaryotic initiator tRNA.

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

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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.

Genome-scale analysis of positional clustering of mouse testis-specific genes

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Lee Bernett TK

2005-01-01

Full Text Available Abstract Background Genes are not randomly distributed on a chromosome as they were thought even after removal of tandem repeats. The positional clustering of co-expressed genes is known in prokaryotes and recently reported in several eukaryotic organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. In order to further investigate the mode of tissue-specific gene clustering in higher eukaryotes, we have performed a genome-scale analysis of positional clustering of the mouse testis-specific genes. Results Our computational analysis shows that a large proportion of testis-specific genes are clustered in groups of 2 to 5 genes in the mouse genome. The number of clusters is much higher than expected by chance even after removal of tandem repeats. Conclusion Our result suggests that testis-specific genes tend to cluster on the mouse chromosomes. This provides another piece of evidence for the hypothesis that clusters of tissue-specific genes do exist.

Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

Science.gov (United States)

Boissy, Robert J; Romberger, Debra J; Roughead, William A; Weissenburger-Moser, Lisa; Poole, Jill A; LeVan, Tricia D

2014-01-01

Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses

Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

Directory of Open Access Journals (Sweden)

Robert J Boissy

Full Text Available Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance. The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70% at the phylum level, Clostridia (44% at the Class level, and Clostridiales at the Order level (41%. In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the

Pyrosequencing analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases.

Science.gov (United States)

Hou, X-L; Cao, Q-Y; Jia, H-Y; Chen, Z

2008-07-01

Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long ( approximately1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 10(4) CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.

Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing.

Science.gov (United States)

Barker, Christopher J; Gillett, Amber; Polkinghorne, Adam; Timms, Peter

2013-12-27

As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut. Copyright © 2013 Elsevier B.V. All rights reserved.

cDNA, genomic sequence cloning and overexpression of ribosomal ...

African Journals Online (AJOL)

RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

Widespread of horizontal gene transfer in the human genome.

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Huang, Wenze; Tsai, Lillian; Li, Yulong; Hua, Nan; Sun, Chen; Wei, Chaochun

2017-04-04

A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome. From the pair-wise alignments between human genome and 53 vertebrate genomes, 1,467 human genome regions (2.6 M bases) from all chromosomes were found to be more conserved with non-mammals than with most mammals. These human genome regions involve 642 known genes, which are enriched with ion binding. Compared to known horizontal gene transfer regions in the human genome, there were few overlapping regions, which indicated horizontal gene transfer is more common than we expected in the human genome. Horizontal gene transfer impacts hundreds of human genes and this study provided insight into potential mechanisms of HGT in the human genome.

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

Science.gov (United States)

Liu, Guozheng; Cao, Dandan; Li, Shuangshuang; Su, Aiguo; Geng, Jianing; Grover, Corrinne E; Hu, Songnian; Hua, Jinping

2013-01-01

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.

Evaluation of culture-based techniques and 454 pyrosequencing for the analysis of fungal diversity in potting media and organic fertilizers.

Science.gov (United States)

Al-Sadi, A M; Al-Mazroui, S S; Phillips, A J L

2015-08-01

Potting media and organic fertilizers (OFs) are commonly used in agricultural systems. However, there is a lack of studies on the efficiency of culture-based techniques in assessing the level of fungal diversity in these products. A study was conducted to investigate the efficiency of seven culture-based techniques and pyrosequencing for characterizing fungal diversity in potting media and OFs. Fungal diversity was evaluated using serial dilution, direct plating and baiting with carrot slices, potato slices, radish seeds, cucumber seeds and cucumber cotyledons. Identity of all the isolates was confirmed on the basis of the internal transcribed spacer region of the ribosomal RNA (ITS rRNA) sequence data. The direct plating technique was found to be superior over other culture-based techniques in the number of fungal species detected. It was also found to be simple and the least time consuming technique. Comparing the efficiency of direct plating with 454 pyrosequencing revealed that pyrosequencing detected 12 and 15 times more fungal species from potting media and OFs respectively. Analysis revealed that there were differences between potting media and OFs in the dominant phyla, classes, orders, families, genera and species detected. Zygomycota (52%) and Chytridiomycota (60%) were the predominant phyla in potting media and OFs respectively. The superiority of pyrosequencing over cultural methods could be related to the ability to detect obligate fungi, slow growing fungi and fungi that exist at low population densities. The evaluated methods in this study, especially direct plating and pyrosequencing, may be used as tools to help detect and reduce movement of unwanted fungi between countries and regions. © 2015 The Society for Applied Microbiology.

Does selection against transcriptional interference shape retroelement-free regions in mammalian genomes?

DEFF Research Database (Denmark)

Mourier, Tobias; Willerslev, Eske

2008-01-01

in generating and maintaining retroelement-free regions in the human genome. METHODOLOGY/PRINCIPAL FINDINGS: Based on the known transcriptional properties of retroelements, we expect long interspersed elements (LINEs) to be able to display a high degree of transcriptional interference. In contrast, we expect......BACKGROUND: Eukaryotic genomes are scattered with retroelements that proliferate through retrotransposition. Although retroelements make up around 40 percent of the human genome, large regions are found to be completely devoid of retroelements. This has been hypothesised to be a result of genomic...... activity of LINEs has been identified previously. CONCLUSIONS/SIGNIFICANCE: Our observations are consistent with the notion that selection against transcriptional interference has contributed to the maintenance and/or generation of retroelement-free regions in the human genome....

David and Goliath: chemical perturbation of eukaryotes by bacteria.

Science.gov (United States)

Ho, Louis K; Nodwell, Justin R

2016-03-01

Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.

Reproduction, symbiosis, and the eukaryotic cell

Science.gov (United States)

Godfrey-Smith, Peter

2015-01-01

This paper develops a conceptual framework for addressing questions about reproduction, individuality, and the units of selection in symbiotic associations, with special attention to the origin of the eukaryotic cell. Three kinds of reproduction are distinguished, and a possible evolutionary sequence giving rise to a mitochondrion-containing eukaryotic cell from an endosymbiotic partnership is analyzed as a series of transitions between each of the three forms of reproduction. The sequence of changes seen in this “egalitarian” evolutionary transition is compared with those that apply in “fraternal” transitions, such as the evolution of multicellularity in animals. PMID:26286983

Comparative transcriptomic analysis of two closely related ground beetle species with marked genital divergence using pyrosequencing.

Science.gov (United States)

Fujimaki, Kotaro; Fujisawa, Tomochika; Yazawa, Shigenobu; Nishimura, Osamu; Sota, Teiji

2014-09-01

Ground beetles of the subgenus Ohomopterus (genus Carabus) show marked divergence in species-specific male and female genital morphologies, which contributes to reproductive isolation among species. Characterizing the genetic basis of species-specific genital morphology is essential for understanding their diversification, but genomic information on Ohomopterus is not yet available. We analyzed mRNA extracted from abdominal sections of the last instar larvae and pupae of two sister species, Carabus (Ohomopterus) iwawakianus and C. (O.) uenoi, which show marked differences in genital morphology, to compare transcriptomic profiles using Roche 454 pyrosequencing. We obtained 1,608,572 high-quality reads and assembled them into 176,278 unique sequences, of which 66,049 sequences were combined into 12,662 clusters. Differential expression analyses for sexed pupae suggested that four and five clusters were differentially expressed between species for males and females, respectively. We also identified orthologous sequences of genes involved in genital development in Drosophila, which potentially affect genital development and species-specific genital morphology in Ohomopterus. This study provides the first large transcriptomic data set for a morphologically diversified beetle group, which can facilitate future studies on the genetic basis of species-specific genitalia.

A novel component of the mitochondrial genome segregation machinery in trypanosomes

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Anneliese Hoffmann

2016-07-01

Full Text Available We recently described a new component (TAC102 of the mitochondrial genome segregation machinery (mtGSM in the protozoan parasite Trypanosoma brucei. T. brucei belongs to a group of organisms that contain a single mitochondrial organelle with a single mitochondrial genome (mt-genome per cell. The mt-genome consists of 5000 minicircles (1 kb and 25 maxicircles (23 kb that are catenated into a large network. After replication of the network its segregation is driven by the separating basal bodies, which are homologous structures to the centrioles organizing the spindle apparatus in many eukaryotes. The structure connecting the basal body to the mt-genome was named the Tripartite Attachment Complex (TAC owing its name to the distribution across three areas in the cell including the two mitochondrial membranes.

Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.

Science.gov (United States)

Reisch, Christopher R; Prather, Kristala L J

2017-01-05

The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A synergism between adaptive effects and evolvability drives whole genome duplication to fixation

NARCIS (Netherlands)

Cuypers, Thomas D; Hogeweg, Paulien; Hogeweg, P.

Whole genome duplication has shaped eukaryotic evolutionary history and has been associated with drastic environmental change and species radiation. While the most common fate of WGD duplicates is a return to single copy, retained duplicates have been found enriched for highly interacting genes.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar (Coleoptera: Curculionidae Reveals Multiple Protease-Like Transcripts.

Directory of Open Access Journals (Sweden)

Arnubio Valencia

Full Text Available The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

Science.gov (United States)

Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair

2016-01-01

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

Genomics and the making of yeast biodiversity.

Science.gov (United States)

Hittinger, Chris Todd; Rokas, Antonis; Bai, Feng-Yan; Boekhout, Teun; Gonçalves, Paula; Jeffries, Thomas W; Kominek, Jacek; Lachance, Marc-André; Libkind, Diego; Rosa, Carlos A; Sampaio, José Paulo; Kurtzman, Cletus P

2015-12-01

Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Megabase replication domains along the human genome: relation to chromatin structure and genome organisation.

Science.gov (United States)

Audit, Benjamin; Zaghloul, Lamia; Baker, Antoine; Arneodo, Alain; Chen, Chun-Long; d'Aubenton-Carafa, Yves; Thermes, Claude

2013-01-01

In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning of the human genome into megabased-size replication domains delineated as N-shaped motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the genome and are bordered by more than 1,000 putative replication origins. We recapitulate the comparison of this partition of the human genome with high-resolution experimental data that confirms that replication domain borders are likely to be preferential replication initiation zones in the germline. In addition, we highlight the specific distribution of experimental and numerical chromatin marks along replication domains. Domain borders correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and around which replication and transcription are highly coordinated. These regions also present a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be linked to local open chromatin fiber state. Altogether, this chapter presents a compartmentalization of the human genome into replication domains that are landmarks of the human genome organization and are likely to play a key role in genome dynamics during evolution and in pathological situations.

The genome of the extremophile crucifer Thellungiella parvula

KAUST Repository

Dassanayake, Maheshi; Oh, Dongha; Haas, Jeffrey S.; Herná ndez, Á lvaro Gonzalez; Hong, Hyewon; Ali, Shahjahan; Yun, Daejin; Bressan, Ray Anthony; Zhu, Jian-Kang; Bohnert, Hans Jü rgen; Cheeseman, John McP

2011-01-01

Thellungiella parvula is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula's extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance. © 2011 Nature America, Inc. All rights reserved.

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

KAUST Repository

Lee, Onon; Wang, Yong; Yang, Jiangke; Lafi, Feras Fawzi; Al-Suwailem, Abdulaziz M.; Qian, Peiyuan

2010-01-01

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus

Large clusters of co-expressed genes in the Drosophila genome.

Science.gov (United States)

Boutanaev, Alexander M; Kalmykova, Alla I; Shevelyov, Yuri Y; Nurminsky, Dmitry I

2002-12-12

Clustering of co-expressed, non-homologous genes on chromosomes implies their co-regulation. In lower eukaryotes, co-expressed genes are often found in pairs. Clustering of genes that share aspects of transcriptional regulation has also been reported in higher eukaryotes. To advance our understanding of the mode of coordinated gene regulation in multicellular organisms, we performed a genome-wide analysis of the chromosomal distribution of co-expressed genes in Drosophila. We identified a total of 1,661 testes-specific genes, one-third of which are clustered on chromosomes. The number of clusters of three or more genes is much higher than expected by chance. We observed a similar trend for genes upregulated in the embryo and in the adult head, although the expression pattern of individual genes cannot be predicted on the basis of chromosomal position alone. Our data suggest that the prevalent mechanism of transcriptional co-regulation in higher eukaryotes operates with extensive chromatin domains that comprise multiple genes.

Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

Science.gov (United States)

Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

2016-04-01

Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

Arsenic and Antimony Transporters in Eukaryotes

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Ewa Maciaszczyk-Dziubinska

2012-03-01

Full Text Available 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.

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. PMID:22489166

Eukaryotic evolutionary transitions are associated with extreme codon bias in functionally-related proteins.

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Nicholas J Hudson

Full Text Available Codon bias in the genome of an organism influences its phenome by changing the speed and efficiency of mRNA translation and hence protein abundance. We hypothesized that differences in codon bias, either between-species differences in orthologous genes, or within-species differences between genes, may play an evolutionary role. To explore this hypothesis, we compared the genome-wide codon bias in six species that occupy vital positions in the Eukaryotic Tree of Life. We acquired the entire protein coding sequences for these organisms, computed the codon bias for all genes in each organism and explored the output for relationships between codon bias and protein function, both within- and between-lineages. We discovered five notable coordinated patterns, with extreme codon bias most pronounced in traits considered highly characteristic of a given lineage. Firstly, the Homo sapiens genome had stronger codon bias for DNA-binding transcription factors than the Saccharomyces cerevisiae genome, whereas the opposite was true for ribosomal proteins--perhaps underscoring transcriptional regulation in the origin of complexity. Secondly, both mammalian species examined possessed extreme codon bias in genes relating to hair--a tissue unique to mammals. Thirdly, Arabidopsis thaliana showed extreme codon bias in genes implicated in cell wall formation and chloroplast function--which are unique to plants. Fourthly, Gallus gallus possessed strong codon bias in a subset of genes encoding mitochondrial proteins--perhaps reflecting the enhanced bioenergetic efficiency in birds that co-evolved with flight. And lastly, the G. gallus genome had extreme codon bias for the Ciliary Neurotrophic Factor--which may help to explain their spontaneous recovery from deafness. We propose that extreme codon bias in groups of genes that encode functionally related proteins has a pathway-level energetic explanation.

Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing.

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Ye, Lin; Zhang, Tong

2011-09-01

This study applied 454 high-throughput pyrosequencing to analyze potentially pathogenic bacteria in activated sludge from 14 municipal wastewater treatment plants (WWTPs) across four countries (China, U.S., Canada, and Singapore), plus the influent and effluent of one of the 14 WWTPs. A total of 370,870 16S rRNA gene sequences with average length of 207 bps were obtained and all of them were assigned to corresponding taxonomic ranks by using RDP classifier and MEGAN. It was found that the most abundant potentially pathogenic bacteria in the WWTPs were affiliated with the genera of Aeromonas and Clostridium. Aeromonas veronii, Aeromonas hydrophila, and Clostridium perfringens were species most similar to the potentially pathogenic bacteria found in this study. Some sequences highly similar (>99%) to Corynebacterium diphtheriae were found in the influent and activated sludge samples from a saline WWTP. Overall, the percentage of the sequences closely related (>99%) to known pathogenic bacteria sequences was about 0.16% of the total sequences. Additionally, a platform-independent Java application (BAND) was developed for graphical visualization of the data of microbial abundance generated by high-throughput pyrosequencing. The approach demonstrated in this study could examine most of the potentially pathogenic bacteria simultaneously instead of one-by-one detection by other methods.

A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

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Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

2013-01-01

SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRi) plasmids | Office of Cancer Genomics

Science.gov (United States)

CTD2 researchers at the University of California in San Francisco developed a modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR/dCas9 system. Catalytically inactive dCas9 enables modular and programmable RNA-guided genome regulation in eukaryotes.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Czech Academy of Sciences Publication Activity Database

Oborník, Miroslav; Kořený, Luděk

2012-01-01

Roč. 492, č. 7427 (2012), s. 59-65 ISSN 0028-0836 Institutional support: RVO:60077344 Keywords : GENE-TRANSFER * BIGELOWIELLA-NATANS * EUKARYOTIC GENOMES * GUILLARDIA-THETA * NUCLEUS * CHLORARACHNIOPHYTE * PROTEINS * SEQUENCE * ORIGIN * CRYPTOPHYTES Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 38.597, year: 2012 http://www.nature.com/nature/journal/v492/n7427/full/nature11681.html

Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units.

Science.gov (United States)

Vettraino, A M; Bonants, P; Tomassini, A; Bruni, N; Vannini, A

2012-11-01

To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs). Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans. Pyrosequencing resulted in 16 965 reads, detecting all species in the template DNA mixture. Reducing the ITS1 sequence identity threshold resulted in a decrease in numbers of unmatched reads but a concomitant increase in the numbers of false MOTUs. The total error rate was 0·63% and comprised mainly mismatches (0·25%) Pyrosequencing of ITS1 region is an efficient and accurate technique for the detection and identification of Phytophthora spp. in environmental samples. However, the risk of allocating false MOTUs, even when demonstrated to be low, may require additional validation with alternative detection methods. Phytophthora spp. are considered among the most destructive groups of invasive plant pathogens, affecting thousands of cultivated and wild plants worldwide. Simultaneous early detection of Phytophthora complexes in environmental samples offers an unique opportunity for the interception of known and unknown species along pathways of introduction, along with the identification of these organisms in invaded environments. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

OpenAIRE

Huerta, Carlos; Borek, Dominika; Machius, Mischa; Grishin, Nick V.; Zhang, Hong

2009-01-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme FAD and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different set of active site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from a pathogenic yeast Candida glabrata...

MIPS: a database for protein sequences and complete genomes.

Science.gov (United States)

Mewes, H W; Hani, J; Pfeiffer, F; Frishman, D

1998-01-01

The MIPS group [Munich Information Center for Protein Sequences of the German National Center for Environment and Health (GSF)] at the Max-Planck-Institute for Biochemistry, Martinsried near Munich, Germany, is involved in a number of data collection activities, including a comprehensive database of the yeast genome, a database reflecting the progress in sequencing the Arabidopsis thaliana genome, the systematic analysis of other small genomes and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database (described elsewhere in this volume). Through its WWW server (http://www.mips.biochem.mpg.de ) MIPS provides access to a variety of generic databases, including a database of protein families as well as automatically generated data by the systematic application of sequence analysis algorithms. The yeast genome sequence and its related information was also compiled on CD-ROM to provide dynamic interactive access to the 16 chromosomes of the first eukaryotic genome unraveled. PMID:9399795

How MCM loading and spreading specify eukaryotic DNA replication initiation sites [version 1; referees: 4 approved

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Olivier Hyrien

2016-08-01

Full Text Available DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs, the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC, they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing

Czech Academy of Sciences Publication Activity Database

Kostovčík, Martin; Bateman, C.C.; Kolařík, Miroslav; Stelinski, L.L.; Jordal, B.H.; Hulcr, J.

2015-01-01

Roč. 9, č. 1 (2015), s. 126-138 ISSN 1751-7362 Institutional support: RVO:61388971 Keywords : ambrosia symbiosis * pyrosequencing Subject RIV: EE - Microbiology, Virology Impact factor: 9.328, year: 2015

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

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Keeling Patrick J

2007-09-01

Full Text Available Abstract Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements

Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

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Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

2014-01-01

ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside

Defensins: antifungal lessons from eukaryotes

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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.

Genome Size Diversity and Its Impact on the Evolution of Land Plants

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Jaume Pellicer

2018-02-01

Full Text Available Genome size is a biodiversity trait that shows staggering diversity across eukaryotes, varying over 64,000-fold. Of all major taxonomic groups, land plants stand out due to their staggering genome size diversity, ranging ca. 2400-fold. As our understanding of the implications and significance of this remarkable genome size diversity in land plants grows, it is becoming increasingly evident that this trait plays not only an important role in shaping the evolution of plant genomes, but also in influencing plant community assemblages at the ecosystem level. Recent advances and improvements in novel sequencing technologies, as well as analytical tools, make it possible to gain critical insights into the genomic and epigenetic mechanisms underpinning genome size changes. In this review we provide an overview of our current understanding of genome size diversity across the different land plant groups, its implications on the biology of the genome and what future directions need to be addressed to fill key knowledge gaps.

KGCAK: a K-mer based database for genome-wide phylogeny and complexity evaluation.

Science.gov (United States)

Wang, Dapeng; Xu, Jiayue; Yu, Jun

2015-09-16

The K-mer approach, treating genomic sequences as simple characters and counting the relative abundance of each string upon a fixed K, has been extensively applied to phylogeny inference for genome assembly, annotation, and comparison. To meet increasing demands for comparing large genome sequences and to promote the use of the K-mer approach, we develop a versatile database, KGCAK ( http://kgcak.big.ac.cn/KGCAK/ ), containing ~8,000 genomes that include genome sequences of diverse life forms (viruses, prokaryotes, protists, animals, and plants) and cellular organelles of eukaryotic lineages. It builds phylogeny based on genomic elements in an alignment-free fashion and provides in-depth data processing enabling users to compare the complexity of genome sequences based on K-mer distribution. We hope that KGCAK becomes a powerful tool for exploring relationship within and among groups of species in a tree of life based on genomic data.

HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

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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.

Next-Generation Genomics Facility at C-CAMP: Accelerating Genomic Research in India

Science.gov (United States)

S, Chandana; Russiachand, Heikham; H, Pradeep; S, Shilpa; M, Ashwini; S, Sahana; B, Jayanth; Atla, Goutham; Jain, Smita; Arunkumar, Nandini; Gowda, Malali

2014-01-01

Next-Generation Sequencing (NGS; http://www.genome.gov/12513162) is a recent life-sciences technological revolution that allows scientists to decode genomes or transcriptomes at a much faster rate with a lower cost. Genomic-based studies are in a relatively slow pace in India due to the non-availability of genomics experts, trained personnel and dedicated service providers. Using NGS there is a lot of potential to study India's national diversity (of all kinds). We at the Centre for Cellular and Molecular Platforms (C-CAMP) have launched the Next Generation Genomics Facility (NGGF) to provide genomics service to scientists, to train researchers and also work on national and international genomic projects. We have HiSeq1000 from Illumina and GS-FLX Plus from Roche454. The long reads from GS FLX Plus, and high sequence depth from HiSeq1000, are the best and ideal hybrid approaches for de novo and re-sequencing of genomes and transcriptomes. At our facility, we have sequenced around 70 different organisms comprising of more than 388 genomes and 615 transcriptomes – prokaryotes and eukaryotes (fungi, plants and animals). In addition we have optimized other unique applications such as small RNA (miRNA, siRNA etc), long Mate-pair sequencing (2 to 20 Kb), Coding sequences (Exome), Methylome (ChIP-Seq), Restriction Mapping (RAD-Seq), Human Leukocyte Antigen (HLA) typing, mixed genomes (metagenomes) and target amplicons, etc. Translating DNA sequence data from NGS sequencer into meaningful information is an important exercise. Under NGGF, we have bioinformatics experts and high-end computing resources to dissect NGS data such as genome assembly and annotation, gene expression, target enrichment, variant calling (SSR or SNP), comparative analysis etc. Our services (sequencing and bioinformatics) have been utilized by more than 45 organizations (academia and industry) both within India and outside, resulting several publications in peer-reviewed journals and several genomic

SINEs as driving forces in genome evolution.

Science.gov (United States)

Schmitz, J

2012-01-01

SINEs are short interspersed elements derived from cellular RNAs that repetitively retropose via RNA intermediates and integrate more or less randomly back into the genome. SINEs propagate almost entirely vertically within their host cells and, once established in the germline, are passed on from generation to generation. As non-autonomous elements, their reverse transcription (from RNA to cDNA) and genomic integration depends on the activity of the enzymatic machinery of autonomous retrotransposons, such as long interspersed elements (LINEs). SINEs are widely distributed in eukaryotes, but are especially effectively propagated in mammalian species. For example, more than a million Alu-SINE copies populate the human genome (approximately 13% of genomic space), and few master copies of them are still active. In the organisms where they occur, SINEs are a challenge to genomic integrity, but in the long term also can serve as beneficial building blocks for evolution, contributing to phenotypic heterogeneity and modifying gene regulatory networks. They substantially expand the genomic space and introduce structural variation to the genome. SINEs have the potential to mutate genes, to alter gene expression, and to generate new parts of genes. A balanced distribution and controlled activity of such properties is crucial to maintaining the organism's dynamic and thriving evolution. Copyright © 2012 S. Karger AG, Basel.

Specificity and evolvability in eukaryotic protein interaction networks.

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Pedro Beltrao

2007-02-01

Full Text Available Progress in uncovering the protein interaction networks of several species has led to questions of what underlying principles might govern their organization. Few studies have tried to determine the impact of protein interaction network evolution on the observed physiological differences between species. Using comparative genomics and structural information, we show here that eukaryotic species have rewired their interactomes at a fast rate of approximately 10(-5 interactions changed per protein pair, per million years of divergence. For Homo sapiens this corresponds to 10(3 interactions changed per million years. Additionally we find that the specificity of binding strongly determines the interaction turnover and that different biological processes show significantly different link dynamics. In particular, human proteins involved in immune response, transport, and establishment of localization show signs of positive selection for change of interactions. Our analysis suggests that a small degree of molecular divergence can give rise to important changes at the network level. We propose that the power law distribution observed in protein interaction networks could be partly explained by the cell's requirement for different degrees of protein binding specificity.

The genome of the extremophile crucifer Thellungiella parvula

KAUST Repository

Dassanayake, Maheshi

2011-08-07

Thellungiella parvula is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula\\'s extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance. © 2011 Nature America, Inc. All rights reserved.

Remembrance of things past retrieved from the Paramecium genome.

Science.gov (United States)

Sperling, Linda

2011-01-01

Paramecium and other ciliates are the only unicellular eukaryotes that separate germinal and somatic functions. A germline micronucleus transmits the genetic information to sexual progeny, while a somatic macronucleus expresses the genetic information during vegetative growth to determine the phenotype. At each sexual generation, a new macronucleus develops from the zygotic nucleus through programmed rearrangements of the germline genome. Paramecium tetraurelia somatic genome sequencing, reviewed here, has provided insight into the organization and evolution of the genome. A series of at least 3 whole genome duplications was detected in the Paramecium lineage and selective pressures that determine the fate of the gene duplicates analyzed. Variability in the somatic DNA was characterized and could be attributed to the genome rearrangement processes. Since, in Paramecium, alternative genome rearrangement patterns can be inherited across sexual generations by homology-dependent epigenetic mechanisms and can affect phenotype, I discuss the possibility that ciliate nuclear dimorphism buffers genetic variation hidden in the germline. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

Evaluation of Automated Ribosomal Intergenic Spacer Analysis for Bacterial Fingerprinting of Rumen Microbiome Compared to Pyrosequencing Technology

Directory of Open Access Journals (Sweden)

Elie Jami

2014-01-01

Full Text Available The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA, an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data.

Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.

Science.gov (United States)

Trama, Jason P; Adelson, Martin E; Mordechai, Eli

2007-12-01

Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. To develop a rapid method for identifying patients infected with MCV via swab sampling. Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

DNA mismatch repair and its many roles in eukaryotic cells

DEFF Research Database (Denmark)

Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

2017-01-01

in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays...... novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore......, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1–independent subpathways of MMR is not known. This review summarizes recent literature...

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

Science.gov (United States)

Christie, Joshua R; Beekman, Madeleine

2017-03-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health.

Science.gov (United States)

Hazkani-Covo, Einat; Martin, William F

2017-05-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

High throughput platforms for structural genomics of integral membrane proteins.

Science.gov (United States)

Mancia, Filippo; Love, James

2011-08-01

Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

Local coexpression domains of two to four genes in the genome of Arabidopsis

NARCIS (Netherlands)

Ren, X.Y.; Fiers, M.W.E.J.; Stiekema, W.J.; Nap, J.P.H.

2005-01-01

Expression of genes in eukaryotic genomes is known to cluster, but cluster size is generally loosely defined and highly variable. We have here taken a very strict definition of cluster as sets of physically adjacent genes that are highly coexpressed and form so-called local coexpression domains. The

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.

Long- and short-term selective forces on malaria parasite genomes

KAUST Repository

Nygaard, Sanne

2010-09-09

Plasmodium parasites, the causal agents of malaria, result in more than 1 million deaths annually. Plasmodium are unicellular eukaryotes with small ~23 Mb genomes encoding ~5200 protein-coding genes. The protein-coding genes comprise about half of these genomes. Although evolutionary processes have a significant impact on malaria control, the selective pressures within Plasmodium genomes are poorly understood, particularly in the non-protein-coding portion of the genome. We use evolutionary methods to describe selective processes in both the coding and non-coding regions of these genomes. Based on genome alignments of seven Plasmodium species, we show that protein-coding, intergenic and intronic regions are all subject to purifying selection and we identify 670 conserved non-genic elements. We then use genome-wide polymorphism data from P. falciparum to describe short-term selective processes in this species and identify some candidate genes for balancing (diversifying) selection. Our analyses suggest that there are many functional elements in the non-genic regions of these genomes and that adaptive evolution has occurred more frequently in the protein-coding regions of the genome. © 2010 Nygaard et al.

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

Science.gov (United States)

Francis, Warren R; Wörheide, Gert

2017-06-01

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Complete mitochondrial genome of Concholepas concholepas inferred by 454 pyrosequencing and mtDNA expression in two mollusc populations.

Science.gov (United States)

Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Gallardo-Escárate, Cristian

2013-03-01

Despite the great relevance of mitochondrial genome analysis in evolutionary studies, there is scarce information on how the transcripts associated with the mitogenome are expressed and their role in the genetic structuring of populations. This work reports the complete mitochondrial genome of the marine gastropod Concholepas concholepas, obtained by 454 pryosequencing, and an analysis of mitochondrial transcripts of two populations 1000 km apart along the Chilean coast. The mitochondrion of C. concholepas is 15,495 base pairs (bp) in size and contains the 37 subunits characteristic of metazoans, as well as a non-coding region of 330 bp. In silico analysis of mitochondrial gene variability showed significant differences among populations. In terms of levels of relative abundance of transcripts associated with mitochondrion in the two populations (assessed by qPCR), the genes associated with complexes III and IV of the mitochondrial genome had the highest levels of expression in the northern population while transcripts associated with the ATP synthase complex had the highest levels of expression in the southern population. Moreover, fifteen polymorphic SNPs were identified in silico between the mitogenomes of the two populations. Four of these markers implied different amino acid substitutions (non-synonymous SNPs). This work contributes novel information regarding the mitochondrial genome structure and mRNA expression levels of C. concholepas. Copyright © 2012 Elsevier Inc. All rights reserved.

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes

Science.gov (United States)

Christie, Joshua R.; Beekman, Madeleine

2017-01-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution. PMID:28025277

Scarless Cas9 Assisted Recombineering (no‐SCAR) in Escherichia coli, an Easy‐to‐Use System for Genome Editing

OpenAIRE

Reisch, Christopher R; Jones, Kristala L.

2018-01-01

The discovery and development of genome editing systems that leverage the site‐specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a “guide” RNA to enable the Cas9 nuclease to make a double‐strand break at a particular genome locus, which is repaired by non‐homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration...

Meeting Report: Towards the Visualization of Genome Activity at Nanoscale Dimensions

International Nuclear Information System (INIS)

Ritland Politz, Joan C.

2006-01-01

A report on the Fifth Annual Nanostructural Genomics meeting, Bar Harbor, USA, 7-10 September 2005. It is a rare meeting where one can hear the latest developments in comparative genome analysis, relate these findings to advances in understanding both the linear and three-dimensional organization of the eukaryotic genome, and see it all beginning to fit into the context of the structure and function of the nucleus, visualized using state-of-the art labeling and microscopic techniques. These cross-disciplinary areas of research have been presented by a diverse group of scientists for the past five years at the Nanostructural Genomics meeting at the Jackson Laboratory in Bar Harbor, and the 2005 meeting again gave attendees much food for thought. In summary, the meeting provided a delightfully unique perspective on the application of exciting experimental breakthroughs at the interface of genomics, cell biology and optical physics.

The sequence and de novo assembly of the giant panda genome

Science.gov (United States)

Li, Ruiqiang; Fan, Wei; Tian, Geng; Zhu, Hongmei; He, Lin; Cai, Jing; Huang, Quanfei; Cai, Qingle; Li, Bo; Bai, Yinqi; Zhang, Zhihe; Zhang, Yaping; Wang, Wen; Li, Jun; Wei, Fuwen; Li, Heng; Jian, Min; Li, Jianwen; Zhang, Zhaolei; Nielsen, Rasmus; Li, Dawei; Gu, Wanjun; Yang, Zhentao; Xuan, Zhaoling; Ryder, Oliver A.; Leung, Frederick Chi-Ching; Zhou, Yan; Cao, Jianjun; Sun, Xiao; Fu, Yonggui; Fang, Xiaodong; Guo, Xiaosen; Wang, Bo; Hou, Rong; Shen, Fujun; Mu, Bo; Ni, Peixiang; Lin, Runmao; Qian, Wubin; Wang, Guodong; Yu, Chang; Nie, Wenhui; Wang, Jinhuan; Wu, Zhigang; Liang, Huiqing; Min, Jiumeng; Wu, Qi; Cheng, Shifeng; Ruan, Jue; Wang, Mingwei; Shi, Zhongbin; Wen, Ming; Liu, Binghang; Ren, Xiaoli; Zheng, Huisong; Dong, Dong; Cook, Kathleen; Shan, Gao; Zhang, Hao; Kosiol, Carolin; Xie, Xueying; Lu, Zuhong; Zheng, Hancheng; Li, Yingrui; Steiner, Cynthia C.; Lam, Tommy Tsan-Yuk; Lin, Siyuan; Zhang, Qinghui; Li, Guoqing; Tian, Jing; Gong, Timing; Liu, Hongde; Zhang, Dejin; Fang, Lin; Ye, Chen; Zhang, Juanbin; Hu, Wenbo; Xu, Anlong; Ren, Yuanyuan; Zhang, Guojie; Bruford, Michael W.; Li, Qibin; Ma, Lijia; Guo, Yiran; An, Na; Hu, Yujie; Zheng, Yang; Shi, Yongyong; Li, Zhiqiang; Liu, Qing; Chen, Yanling; Zhao, Jing; Qu, Ning; Zhao, Shancen; Tian, Feng; Wang, Xiaoling; Wang, Haiyin; Xu, Lizhi; Liu, Xiao; Vinar, Tomas; Wang, Yajun; Lam, Tak-Wah; Yiu, Siu-Ming; Liu, Shiping; Zhang, Hemin; Li, Desheng; Huang, Yan; Wang, Xia; Yang, Guohua; Jiang, Zhi; Wang, Junyi; Qin, Nan; Li, Li; Li, Jingxiang; Bolund, Lars; Kristiansen, Karsten; Wong, Gane Ka-Shu; Olson, Maynard; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

2013-01-01

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes. PMID:20010809

Molecular fossils in modern genomes provide physiological and geochemical insights to the ancient earth (Invited)

Science.gov (United States)

Dupont, C.; Caetano-Anolles, G.

2010-12-01

The genomes of extant organisms are ultimately derived from ancient life, thus theoretically contain insight to ancient physiology, ecology, and environments. In particular, metalloenzymes may be particularly insightful. The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic pro- teomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predomi- nantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, pro- viding further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology, and Zn bioavailabil- ity may have been a limiting factor in eukaryotic evolution. The results presented here provide an evolutionary timeline based on genomic characteristics, and key hypotheses can be tested by alternative geochemical methods.

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

Science.gov (United States)

Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

2016-01-01

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

Exploration of the Germline Genome of the Ciliate Chilodonella uncinata through Single-Cell Omics (Transcriptomics and Genomics

Directory of Open Access Journals (Sweden)

Xyrus X. Maurer-Alcalá

2018-01-01

Full Text Available Separate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera. In ciliates, germline-limited (i.e., micronuclear-specific DNA is eliminated during the development of a new somatic (i.e., macronuclear genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliates Oxytricha trifallax, Paramecium tetraurelia, and Tetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of “purified” germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliate Chilodonella uncinata, a member of the understudied class Phyllopharyngea. Our analyses reveal the following: (i large gene families contain a disproportionate number of genes from scrambled germline loci; (ii germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.

Gonococcal attachment to eukaryotic cells

International Nuclear Information System (INIS)

James, J.F.; Lammel, C.J.; Draper, D.L.; Brown, D.A.; Sweet, R.L.; Brooks, G.F.

1983-01-01

The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with [ 3 H]- and [ 14 C]adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, HeLa 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants from transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

Energy Technology Data Exchange (ETDEWEB)

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

QuartetS-DB: a large-scale orthology database for prokaryotes and eukaryotes inferred by evolutionary evidence

Directory of Open Access Journals (Sweden)

Yu Chenggang

2012-06-01

Full Text Available Abstract Background The concept of orthology is key to decoding evolutionary relationships among genes across different species using comparative genomics. QuartetS is a recently reported algorithm for large-scale orthology detection. Based on the well-established evolutionary principle that gene duplication events discriminate paralogous from orthologous genes, QuartetS has been shown to improve orthology detection accuracy while maintaining computational efficiency. Description QuartetS-DB is a new orthology database constructed using the QuartetS algorithm. The database provides orthology predictions among 1621 complete genomes (1365 bacterial, 92 archaeal, and 164 eukaryotic, covering more than seven million proteins and four million pairwise orthologs. It is a major source of orthologous groups, containing more than 300,000 groups of orthologous proteins and 236,000 corresponding gene trees. The database also provides over 500,000 groups of inparalogs. In addition to its size, a distinguishing feature of QuartetS-DB is the ability to allow users to select a cutoff value that modulates the balance between prediction accuracy and coverage of the retrieved pairwise orthologs. The database is accessible at https://applications.bioanalysis.org/quartetsdb. Conclusions QuartetS-DB is one of the largest orthology resources available to date. Because its orthology predictions are underpinned by evolutionary evidence obtained from sequenced genomes, we expect its accuracy to continue to increase in future releases as the genomes of additional species are sequenced.

Bacterial flora-typing with targeted, chip-based Pyrosequencing

Directory of Open Access Journals (Sweden)

El-Sayed Yasser Y

2007-11-01

Full Text Available Abstract Background The metagenomic analysis of microbial communities holds the potential to improve our understanding of the role of microbes in clinical conditions. Recent, dramatic improvements in DNA sequencing throughput and cost will enable such analyses on individuals. However, such advances in throughput generally come at the cost of shorter read-lengths, limiting the discriminatory power of each read. In particular, classifying the microbial content of samples by sequencing the Results We describe a method for identifying the phylogenetic content of bacterial samples using high-throughput Pyrosequencing targeted at the 16S rRNA gene. Our analysis is adapted to the shorter read-lengths of such technology and uses a database of 16S rDNA to determine the most specific phylogenetic classification for reads, resulting in a weighted phylogenetic tree characterizing the content of the sample. We present results for six samples obtained from the human vagina during pregnancy that corroborates previous studies using conventional techniques. Next, we analyze the power of our method to classify reads at each level of the phylogeny using simulation experiments. We assess the impacts of read-length and database completeness on our method, and predict how we do as technology improves and more bacteria are sequenced. Finally, we study the utility of targeting specific 16S variable regions and show that such an approach considerably improves results for certain types of microbial samples. Using simulation, our method can be used to determine the most informative variable region. Conclusion This study provides positive validation of the effectiveness of targeting 16S metagenomes using short-read sequencing technology. Our methodology allows us to infer the most specific assignment of the sequence reads within the phylogeny, and to identify the most discriminative variable region to target. The analysis of high-throughput Pyrosequencing on human flora

Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

Science.gov (United States)

Dennig, Jörg

Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

Population Genomics of Paramecium Species.

Science.gov (United States)

Johri, Parul; Krenek, Sascha; Marinov, Georgi K; Doak, Thomas G; Berendonk, Thomas U; Lynch, Michael

2017-05-01

Population-genomic analyses are essential to understanding factors shaping genomic variation and lineage-specific sequence constraints. The dearth of such analyses for unicellular eukaryotes prompted us to assess genomic variation in Paramecium, one of the most well-studied ciliate genera. The Paramecium aurelia complex consists of ∼15 morphologically indistinguishable species that diverged subsequent to two rounds of whole-genome duplications (WGDs, as long as 320 MYA) and possess extremely streamlined genomes. We examine patterns of both nuclear and mitochondrial polymorphism, by sequencing whole genomes of 10-13 worldwide isolates of each of three species belonging to the P. aurelia complex: P. tetraurelia, P. biaurelia, P. sexaurelia, as well as two outgroup species that do not share the WGDs: P. caudatum and P. multimicronucleatum. An apparent absence of global geographic population structure suggests continuous or recent dispersal of Paramecium over long distances. Intergenic regions are highly constrained relative to coding sequences, especially in P. caudatum and P. multimicronucleatum that have shorter intergenic distances. Sequence diversity and divergence are reduced up to ∼100-150 bp both upstream and downstream of genes, suggesting strong constraints imposed by the presence of densely packed regulatory modules. In addition, comparison of sequence variation at non-synonymous and synonymous sites suggests similar recent selective pressures on paralogs within and orthologs across the deeply diverging species. This study presents the first genome-wide population-genomic analysis in ciliates and provides a valuable resource for future studies in evolutionary and functional genetics in Paramecium. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High-Resolution Replication Profiles Define the Stochastic Nature of Genome Replication Initiation and Termination

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Michelle Hawkins

2013-11-01

Full Text Available Eukaryotic genome replication is stochastic, and each cell uses a different cohort of replication origins. We demonstrate that interpreting high-resolution Saccharomyces cerevisiae genome replication data with a mathematical model allows quantification of the stochastic nature of genome replication, including the efficiency of each origin and the distribution of termination events. Single-cell measurements support the inferred values for stochastic origin activation time. A strain, in which three origins were inactivated, confirmed that the distribution of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

Science.gov (United States)

Debode, Frederic; Janssen, Eric; Bragard, Claude; Berben, Gilbert

2017-08-01

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

Elevated Rate of Genome Rearrangements in Radiation-Resistant Bacteria.

Science.gov (United States)

Repar, Jelena; Supek, Fran; Klanjscek, Tin; Warnecke, Tobias; Zahradka, Ksenija; Zahradka, Davor

2017-04-01

A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation-resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compensate for the genome-destabilizing effect of environmental DNA damage and may be expected to result in a more conserved gene order in radiation-resistant species. However, here we show that rates of genome rearrangements, measured as loss of gene order conservation with time, are higher in radiation-resistant species in multiple, phylogenetically independent groups of bacteria. Comparison of indicators of selection for genome organization between radiation-resistant and phylogenetically matched, nonresistant species argues against tolerance to disruption of genome structure as a strategy for radiation resistance. Interestingly, an important mechanism affecting genome rearrangements in prokaryotes, the symmetrical inversions around the origin of DNA replication, shapes genome structure of both radiation-resistant and nonresistant species. In conclusion, the opposing effects of environmental DNA damage and DNA repair result in elevated rates of genome rearrangements in radiation-resistant bacteria. Copyright © 2017 Repar et al.

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii.

Science.gov (United States)

Di Rienzi, Sara C; Lindstrom, Kimberly C; Lancaster, Ragina; Rolczynski, Lisa; Raghuraman, M K; Brewer, Bonita J

2011-02-01

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution. 2010 John Wiley & Sons, Ltd.

The current state of eukaryotic DNA base damage and repair.

Science.gov (United States)

Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

2015-12-02

DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nuclear envelope and genome interactions in cell fate

Science.gov (United States)

Talamas, Jessica A.; Capelson, Maya

2015-01-01

The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

[Compartmentalization of the cell nucleus and spatial organization of the genome].

Science.gov (United States)

Gavrilov, A A; Razin, S V

2015-01-01

The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

Nothing in Evolution Makes Sense Except in the Light of Genomics: Read–Write Genome Evolution as an Active Biological Process

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James A. Shapiro

2016-06-01

Full Text Available The 21st century genomics-based analysis of evolutionary variation reveals a number of novel features impossible to predict when Dobzhansky and other evolutionary biologists formulated the neo-Darwinian Modern Synthesis in the middle of the last century. These include three distinct realms of cell evolution; symbiogenetic fusions forming eukaryotic cells with multiple genome compartments; horizontal organelle, virus and DNA transfers; functional organization of proteins as systems of interacting domains subject to rapid evolution by exon shuffling and exonization; distributed genome networks integrated by mobile repetitive regulatory signals; and regulation of multicellular development by non-coding lncRNAs containing repetitive sequence components. Rather than single gene traits, all phenotypes involve coordinated activity by multiple interacting cell molecules. Genomes contain abundant and functional repetitive components in addition to the unique coding sequences envisaged in the early days of molecular biology. Combinatorial coding, plus the biochemical abilities cells possess to rearrange DNA molecules, constitute a powerful toolbox for adaptive genome rewriting. That is, cells possess “Read–Write Genomes” they alter by numerous biochemical processes capable of rapidly restructuring cellular DNA molecules. Rather than viewing genome evolution as a series of accidental modifications, we can now study it as a complex biological process of active self-modification.

Eelgrass Leaf Surface Microbiomes Are Locally Variable and Highly Correlated with Epibiotic Eukaryotes

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Mia M. Bengtsson

2017-07-01

Full Text Available Eelgrass (Zostera marina is a marine foundation species essential for coastal ecosystem services around the northern hemisphere. Like all macroscopic organisms, it possesses a microbiome (here defined as an associated prokaryotic community which may play critical roles in modulating the interaction of eelgrass with its environment. For example, its leaf surface microbiome could inhibit or attract eukaryotic epibionts which may overgrow the eelgrass leading to reduced primary productivity and subsequent eelgrass meadow decline. We used amplicon sequencing of the 16S and 18S rRNA genes of prokaryotes and eukaryotes to assess the leaf surface microbiome (prokaryotes as well as eukaryotic epibionts in- and outside lagoons on the German Baltic Sea coast. Prokaryote microbiomes varied substantially both between sites inside lagoons and between open coastal and lagoon sites. Water depth, leaf area and biofilm chlorophyll a concentration explained a large amount of variation in both prokaryotic and eukaryotic community composition. The prokaryotic microbiome and eukaryotic epibiont communities were highly correlated, and network analysis revealed disproportionate co-occurrence between a limited number of eukaryotic taxa and several bacterial taxa. This suggests that eelgrass leaf surfaces are home to a mosaic of microbiomes of several epibiotic eukaryotes, in addition to the microbiome of the eelgrass itself. Our findings thereby underline that eukaryotic diversity should be taken into account in order to explain prokaryotic microbiome assembly and dynamics in aquatic environments.

GENEPEASE Genomic tools for assessment of pesticide effects on the agricultural soil ecosystem

DEFF Research Database (Denmark)

Jacobsen, Carsten Suhr; Feld, Louise; Hjelmsø, Mathis Hjort

The project focussed on validating RNA based methods as potential genomic tools in assessment of agricultural soil ecosystems. It was shown that the mRNA based technique was very sensitive and the effects was seen in the same situations as when the OECD nitrification assay showed an effect. 16S r......RNA based pyrosequencing of bacterial communities in soil was shown to report different than just DNA based analysis and indicated unlike the DNA measurement that the community was developing. Finally microarray analysis was compared to traditional test for toxicity testing of Folsomia candida and showed...

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

Science.gov (United States)

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Development and characterization of microsatellite markers from the humivorous termite Cavitermes tuberosus (Isoptera: Termitinae) using pyrosequencing technology

Czech Academy of Sciences Publication Activity Database

Fournier, D.; Hanus, Robert; Roisin, Y.

2015-01-01

Roč. 7, č. 2 (2015), s. 521-524 ISSN 1877-7252 Institutional support: RVO:61388963 Keywords : Cavitermes tuberosus * termite * microsatellite * pyrosequencing * population genetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.446, year: 2015

Interstitial telomere-like repeats in the Arabidopsis thaliana genome.

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Uchida, Wakana; Matsunaga, Sachihiro; Sugiyama, Ryuji; Kawano, Shigeyuki

2002-02-01

Eukaryotic chromosomal ends are protected by telomeres, which are thought to play an important role in ensuring the complete replication of chromosomes. On the other hand, non-functional telomere-like repeats in the interchromosomal regions (interstitial telomeric repeats; ITRs) have been reported in several eukaryotes. In this study, we identified eight ITRs in the Arabidopsis thaliana genome, each consisting of complete and degenerate 300- to 1200-bp sequences. The ITRs were grouped into three classes (class IA-B, class II, and class IIIA-E) based on the degeneracy of the telomeric repeats in ITRs. The telomeric repeats of the two ITRs in class I were conserved for the most part, whereas the single ITR in class II, and the five ITRs in class III were relatively degenerated. In addition, degenerate ITRs were surrounded by common sequences that shared 70-100% homology to each other; these are named ITR-adjacent sequences (IAS). Although the genomic regions around ITRs in class I lacked IAS, those around ITRs in class II contained IAS (IASa), and those around five ITRs in class III had nine types of IAS (IASb, c, d, e, f, g, h, i, and j). Ten IAS types in classes II and III showed no significant homology to each other. The chromosomal locations of ITRs and IAS were not category-related, but most of them were adjacent to, or part of, a centromere. These results show that the A. thaliana genome has undergone chromosomal rearrangements, such as end-fusions and segmental duplications.

Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

DEFF Research Database (Denmark)

2018-01-01

The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryot...

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack.

Science.gov (United States)

Fügi, Matthias A; Gunasekera, Kapila; Ochsenreiter, Torsten; Guan, Xueli; Wenk, Markus R; Mäser, Pascal

2014-05-01

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Elevated Rate of Genome Rearrangements in Radiation-Resistant Bacteria

OpenAIRE

Repar, Jelena; Supek, Fran; Klanjscek, Tin; Warnecke, Tobias; Zahradka, Ksenija; Zahradka, Davor

2017-01-01

A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation-resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compen...

Widespread of horizontal gene transfer in the human genome

OpenAIRE

Huang, Wenze; Tsai, Lillian; Li, Yulong; Hua, Nan; Sun, Chen; Wei, Chaochun

2017-01-01

Background A fundamental concept in biology is that heritable material is passed from parents to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic materials between different species. Horizontal gene transfer has been found prevalent in prokaryotes but very rare in eukaryote. In this paper, we investigate horizontal gene transfer in the human genome. Results From the pa...

Inorganic phosphate uptake in unicellular eukaryotes.

Science.gov (United States)

Dick, Claudia F; Dos-Santos, André L A; Meyer-Fernandes, José R

2014-07-01

Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field. Copyright © 2014 Elsevier B.V. All rights reserved.

Uniting sex and eukaryote origins in an emerging oxygenic world.

Science.gov (United States)

Gross, Jeferson; Bhattacharya, Debashish

2010-08-23

Theories about eukaryote origins (eukaryogenesis) need to provide unified explanations for the emergence of diverse complex features that define this lineage. Models that propose a prokaryote-to-eukaryote transition are gridlocked between the opposing "phagocytosis first" and "mitochondria as seed" paradigms, neither of which fully explain the origins of eukaryote cell complexity. Sex (outcrossing with meiosis) is an example of an elaborate trait not yet satisfactorily addressed in theories about eukaryogenesis. The ancestral nature of meiosis and its dependence on eukaryote cell biology suggest that the emergence of sex and eukaryogenesis were simultaneous and synergic and may be explained by a common selective pressure. We propose that a local rise in oxygen levels, due to cyanobacterial photosynthesis in ancient Archean microenvironments, was highly toxic to the surrounding biota. This selective pressure drove the transformation of an archaeal (archaebacterial) lineage into the first eukaryotes. Key is that oxygen might have acted in synergy with environmental stresses such as ultraviolet (UV) radiation and/or desiccation that resulted in the accumulation of reactive oxygen species (ROS). The emergence of eukaryote features such as the endomembrane system and acquisition of the mitochondrion are posited as strategies to cope with a metabolic crisis in the cell plasma membrane and the accumulation of ROS, respectively. Selective pressure for efficient repair of ROS/UV-damaged DNA drove the evolution of sex, which required cell-cell fusions, cytoskeleton-mediated chromosome movement, and emergence of the nuclear envelope. Our model implies that evolution of sex and eukaryogenesis were inseparable processes. Several types of data can be used to test our hypothesis. These include paleontological predictions, simulation of ancient oxygenic microenvironments, and cell biological experiments with Archaea exposed to ROS and UV stresses. Studies of archaeal conjugation

Sex is a ubiquitous, ancient, and inherent attribute of eukaryotic life

NARCIS (Netherlands)

Speijer, Dave; Lukeš, Julius; Eliáš, Marek

2015-01-01

Sexual reproduction and clonality in eukaryotes are mostly seen as exclusive, the latter being rather exceptional. This view might be biased by focusing almost exclusively on metazoans. We analyze and discuss reproduction in the context of extant eukaryotic diversity, paying special attention to

Evaluation of Mammalian Interspersed Repeats to investigate the goat genome

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P. Mariani

2010-01-01

Full Text Available Among the repeated sequences present in most eukaryotic genomes, SINEs (Short Interspersed Nuclear Elements are widely used to investigate evolution in the mammalian order (Buchanan et al., 1999. One family of these repetitive sequences, the MIR (Mammalian Interspersed Repeats; Jurka et al., 1995, is ubiquitous in all mammals.MIR elements are tRNA-derived SINEs and are identifiable by a conserved core region of about 70 nucleotides.

Enzymes from Higher Eukaryotes for Industrial Biocatalysis

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Zhibin Liu

2004-01-01

Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

Evolution of DNA replication protein complexes in eukaryotes and Archaea.

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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.

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Energy Technology Data Exchange (ETDEWEB)

Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

2010-05-07

Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Directory of Open Access Journals (Sweden)

Tran Duc

2010-05-01

Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

Science.gov (United States)

Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

2015-03-05

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients.

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Ibrahim Hamad

Full Text Available Research on the relationship between changes in the gut microbiota and human disease, including AIDS, is a growing field. However, studies on the eukaryotic component of the intestinal microbiota have just begun and have not yet been conducted in HIV-infected patients. Moreover, eukaryotic community profiling is influenced by the use of different methodologies at each step of culture-independent techniques. Herein, initially, four DNA extraction protocols were compared to test the efficiency of each method in recovering eukaryotic DNA from fecal samples. Our results revealed that recovering eukaryotic components from fecal samples differs significantly among DNA extraction methods. Subsequently, the composition of the intestinal eukaryotic microbiota was evaluated in HIV-infected patients and healthy volunteers through clone sequencing, high-throughput sequencing of nuclear ribosomal internal transcribed spacers 1 (ITS1 and 2 (ITS2 amplicons and real-time PCRs. Our results revealed that not only richness (Chao-1 index and alpha diversity (Shannon diversity differ between HIV-infected patients and healthy volunteers, depending on the molecular strategy used, but also the global eukaryotic community composition, with little overlapping taxa found between techniques. Moreover, our results based on cloning libraries and ITS1/ITS2 metabarcoding sequencing showed significant differences in fungal composition between HIV-infected patients and healthy volunteers, but without distinct clusters separating the two groups. Malassezia restricta was significantly more prevalent in fecal samples of HIV-infected patients, according to cloning libraries, whereas operational taxonomic units (OTUs belonging to Candida albicans and Candida tropicalis were significantly more abundant in fecal samples of HIV-infected patients compared to healthy subjects in both ITS subregions. Finally, real-time PCR showed the presence of Microsporidia, Giardia lamblia, Blastocystis

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

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Cui Hongli

2012-11-01

Full Text Available Abstract Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS. PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic

Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

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Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

2012-11-16

Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins

The genome of the diatom Thalassiosira pseudonana: Ecology,evolution, and metabolism

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Ambrust, E.V.; Berges, J.; Bowler, C.; Green, B.; Martinez, D.; Putnam, N.; Zhou, S.; Allen, A.; Apt, K.; Bechner, M.; Brzezinski, M.; Chaal, B.; Chiovitti, A.; Davis, A.; Goodstein, D.; Hadi, M.; Hellsten,U.; Hildebrand, M.; Jenkins, B.; Jurka, J.; Kapitonov, V.; Kroger, N.; Lau, W.; Lane, T.; Larimer, F.; Lippmeier, J.; Lucas, S.; Medina, M.; Montsant, A.; Obornik, M.; Parker, M. Schnitzler; Palenik, B.; Pazour,G.; Richardson, P.; Rynearson, T.; Saito, M.; Schwartz, D.; Thamatrakoln,K.; Valentin, K.; Vardi, A.; Wilkerson, F.; Rokhsar, D.; Vardi, A.; Wilkerson, F.P.; Rokhsar, D.S.

2004-09-01

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for {approx}20% of global carbon fixation. We report the 34 Mbp draft nuclear genome of the marine diatom, Thalassiosira pseudonana and its 129 Kbp plastid and 44 Kbp mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, utilization of a range of nitrogenous compounds and a complete urea cycle, all attributes that allow diatoms to prosper in the marine environment. Diatoms are unicellular, photosynthetic, eukaryotic algae found throughout the world's oceans and freshwater systems. They form the base of short, energetically-efficient food webs that support large-scale coastal fisheries. Photosynthesis by marine diatoms generates as much as 40% of the 45-50 billion tonnes of organic carbon produced each year in the sea (1), and their role in global carbon cycling is predicted to be comparable to that of all terrestrial rainforests combined (2, 3). Over geological time, diatoms may have influenced global climate by changing the flux of atmospheric carbon dioxide into the oceans (4). A defining feature of diatoms is their ornately patterned silicified cell wall or frustule, which displays species-specific nano-structures of such fine detail that diatoms have long been used to test the resolution of optical microscopes. Recent attention has focused on biosynthesis of these nano-structures as a paradigm for future silica nanotechnology (5). The long history (over 180 million years) and dominance of diatoms in the oceans is reflected by their contributions to vast deposits of diatomite, most cherts and a significant fraction of current petroleum reserves (6). As photosynthetic heterokonts, diatoms reflect a fundamentally

Exceptionally high levels of recombination across the honey bee genome.

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Beye, Martin; Gattermeier, Irene; Hasselmann, Martin; Gempe, Tanja; Schioett, Morten; Baines, John F; Schlipalius, David; Mougel, Florence; Emore, Christine; Rueppell, Olav; Sirviö, Anu; Guzmán-Novoa, Ernesto; Hunt, Greg; Solignac, Michel; Page, Robert E

2006-11-01

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

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Gerda Saxer

Full Text Available Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10(-9, with a Poisson confidence interval of 4.1×10(-9 - 9.5×10(-9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10(-11, with a Poisson confidence interval ranging from 7.4×10(-13 to 1.6×10(-10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania

Czech Academy of Sciences Publication Activity Database

Flegontov, P.; Butenko, A.; Firsov, Sergei; Kraeva, N.; Eliáš, M.; Field, M.C.; Filatov, D.; Flegontova, Olga; Gerasimov, E. S.; Hlaváčová, J.; Ishemgulova, A.; Jackson, A. P.; Kelly, S.; Kostygov, A.Y.; Logacheva, M.D.; Maslov, D. A.; Opperdoes, F. R.; O'Reilly, A.; Sádlová, J.; Ševčíková, T.; Venkatesh, D.; Vlček, Čestmír; Volf, P.; Votýpka, Jan; Záhonová, K.; Yurchenko, V.; Lukeš, Julius

2016-01-01

Roč. 6, MAR 29 (2016), č. článku 23704. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA14-23986S; GA ČR GA13-24983S EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 ; RVO:68378050 Keywords : amino-acid sites * leader rna gene * insect trypanosomatids * phylogenetic analysis * functional genomics * eukaryotic genomes * positive selection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.259, year: 2016

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

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Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S

2016-05-01

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Recognition of extremophilic archaeal viruses by eukaryotic cells: a promising nanoplatform from the third domain of life

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Uldahl, Kristine Buch; Wu, Linping; Hall, Arnaldur; Papathanasiou, Pavlos; Peng, Xu; Moghimi, Seyed Moein

2016-01-01

Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration of novel medical nanoplatforms. Here, we selected two archaeal viruses Sulfolobus monocaudavirus 1 (SMV1) and Sulfolobus spindle shaped virus 2 (SSV2) owing to their unique spindle shape, hyperthermostable and acid-resistant nature and studied their interaction with mammalian cells. Accordingly, we followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path for selective cell targeting. On internalization, both viruses localize to the lysosomal compartments. Neither SMV1, nor SSV2 induced any detrimental effect on cell morphology, plasma membrane and mitochondrial functionality. This is the first study demonstrating recognition of archaeal viruses by eukaryotic cells which provides good basis for future exploration of archaeal viruses in bioengineering and development of multifunctional vectors. PMID:27892499

Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

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Gillet Laurent

2011-08-01

Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC, the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA. As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G differs from the other prDNA units (prDNA-inner. Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

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Yi-Mo Deng

Full Text Available BACKGROUND: Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. METHODOLOGY/PRINCIPAL FINDINGS: A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. CONCLUSIONS/SIGNIFICANCE: In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

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Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G

2011-01-01

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

ProteinHistorian: tools for the comparative analysis of eukaryote protein origin.

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John A Capra

Full Text Available The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are

The independent prokaryotic origins of eukaryotic fructose-1, 6-bisphosphatase and sedoheptulose-1, 7-bisphosphatase and the implications of their origins for the evolution of eukaryotic Calvin cycle

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Jiang Yong-Hai

2012-10-01

Full Text Available Abstract Background In the Calvin cycle of eubacteria, the dephosphorylations of both fructose-1, 6-bisphosphate (FBP and sedoheptulose-1, 7-bisphosphate (SBP are catalyzed by the same bifunctional enzyme: fructose-1, 6-bisphosphatase/sedoheptulose-1, 7-bisphosphatase (F/SBPase, while in that of eukaryotic chloroplasts by two distinct enzymes: chloroplastic fructose-1, 6-bisphosphatase (FBPase and sedoheptulose-1, 7-bisphosphatase (SBPase, respectively. It was proposed that these two eukaryotic enzymes arose from the divergence of a common ancestral eubacterial bifunctional F/SBPase of mitochondrial origin. However, no specific affinity between SBPase and eubacterial FBPase or F/SBPase can be observed in the previous phylogenetic analyses, and it is hard to explain why SBPase and/or F/SBPase are/is absent from most extant nonphotosynthetic eukaryotes according to this scenario. Results Domain analysis indicated that eubacterial F/SBPase of two different resources contain distinct domains: proteobacterial F/SBPases contain typical FBPase domain, while cyanobacterial F/SBPases possess FBPase_glpX domain. Therefore, like prokaryotic FBPase, eubacterial F/SBPase can also be divided into two evolutionarily distant classes (Class I and II. Phylogenetic analysis based on a much larger taxonomic sampling than previous work revealed that all eukaryotic SBPase cluster together and form a close sister group to the clade of epsilon-proteobacterial Class I FBPase which are gluconeogenesis-specific enzymes, while all eukaryotic chloroplast FBPase group together with eukaryotic cytosolic FBPase and form another distinct clade which then groups with the Class I FBPase of diverse eubacteria. Motif analysis of these enzymes also supports these phylogenetic correlations. Conclusions There are two evolutionarily distant classes of eubacterial bifunctional F/SBPase. Eukaryotic FBPase and SBPase do not diverge from either of them but have two independent origins

Eukaryotic ribosome display with in situ DNA recovery.

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He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing.

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Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie

2010-10-01

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

Draft genome sequence of Cercospora sojina isolate S9, a fungus causing frogeye leaf spot (FLS disease of soybean

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Fanchang Zeng

2017-06-01

Full Text Available Fungi are the causal agents of many of the world's most serious plant diseases causing disastrous consequences for large-scale agricultural production. Pathogenicity genomic basis is complex in fungi as multicellular eukaryotic pathogens. The fungus Cercospora sojina is a plant pathogen that threatens global soybean supplies. Here, we report the genome sequence of C. sojina strain S9 and detect genome features and predicted genomic elements. The genome sequence of C. sojina is a valuable resource with potential in studying the fungal pathogenicity and soybean host resistance to frogeye leaf spot (FLS, which is caused by C. sojina. The C. sojina genome sequence has been deposited and available at DDBJ/EMBL/GenBank under the project accession number AHPQ00000000.

Mitochondrial uncoupling proteins in unicellular eukaryotes.

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Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

2010-01-01

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

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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.

Impact of nuclear organization and chromatin structure on DNA repair and genome stability

International Nuclear Information System (INIS)

Batte, Amandine

2016-01-01

The non-random organization of the eukaryotic cell nucleus and the folding of genome in chromatin more or less condensed can influence many functions related to DNA metabolism, including genome stability. Double-strand breaks (DSBs) are the most deleterious DNA damages for the cells. To preserve genome integrity, eukaryotic cells thus developed DSB repair mechanisms conserved from yeast to human, among which homologous recombination (HR) that uses an intact homologous sequence to repair a broken chromosome. HR can be separated in two sub-pathways: Gene Conversion (GC) transfers genetic information from one molecule to its homologous and Break Induced Replication (BIR) establishes a replication fork than can proceed until the chromosome end. My doctorate work was focused on the contribution of the chromatin context and 3D genome organization on DSB repair. In S. cerevisiae, nuclear organization and heterochromatin spreading at sub-telomeres can be modified through the overexpression of the Sir3 or sir3A2Q mutant proteins. We demonstrated that reducing the physical distance between homologous sequences increased GC rates, reinforcing the notion that homology search is a limiting step for recombination. We also showed that hetero-chromatinization of DSB site fine-tunes DSB resection, limiting the loss of the DSB ends required to perform homology search and complete HR. Finally, we noticed that the presence of heterochromatin at the donor locus decreased both GC and BIR efficiencies, probably by affecting strand invasion. This work highlights new regulatory pathways of DNA repair. (author) [fr

CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

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Xuelian Wang

2016-01-01

Full Text Available Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic.

Draft genome of the gayal, Bos frontalis

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Wang, Ming-Shan; Zeng, Yan; Wang, Xiao; Nie, Wen-Hui; Wang, Jin-Huan; Su, Wei-Ting; Xiong, Zi-Jun; Wang, Sheng; Qu, Kai-Xing; Yan, Shou-Qing; Yang, Min-Min; Wang, Wen; Dong, Yang; Zhang, Ya-Ping

2017-01-01

Abstract Gayal (Bos frontalis), also known as mithan or mithun, is a large endangered semi-domesticated bovine that has a limited geographical distribution in the hill-forests of China, Northeast India, Bangladesh, Myanmar, and Bhutan. Many questions about the gayal such as its origin, population history, and genetic basis of local adaptation remain largely unresolved. De novo sequencing and assembly of the whole gayal genome provides an opportunity to address these issues. We report a high-depth sequencing, de novo assembly, and annotation of a female Chinese gayal genome. Based on the Illumina genomic sequencing platform, we have generated 350.38 Gb of raw data from 16 different insert-size libraries. A total of 276.86 Gb of clean data is retained after quality control. The assembled genome is about 2.85 Gb with scaffold and contig N50 sizes of 2.74 Mb and 14.41 kb, respectively. Repetitive elements account for 48.13% of the genome. Gene annotation has yielded 26 667 protein-coding genes, of which 97.18% have been functionally annotated. BUSCO assessment shows that our assembly captures 93% (3183 of 4104) of the core eukaryotic genes and 83.1% of vertebrate universal single-copy orthologs. We provide the first comprehensive de novo genome of the gayal. This genetic resource is integral for investigating the origin of the gayal and performing comparative genomic studies to improve understanding of the speciation and divergence of bovine species. The assembled genome could be used as reference in future population genetic studies of gayal. PMID:29048483

Quantitative prediction of shrimp disease incidence via the profiles of gut eukaryotic microbiota.

Science.gov (United States)

Xiong, Jinbo; Yu, Weina; Dai, Wenfang; Zhang, Jinjie; Qiu, Qiongfen; Ou, Changrong

2018-04-01

One common notion is emerging that gut eukaryotes are commensal or beneficial, rather than detrimental. To date, however, surprisingly few studies have been taken to discern the factors that govern the assembly of gut eukaryotes, despite growing interest in the dysbiosis of gut microbiota-disease relationship. Herein, we firstly explored how the gut eukaryotic microbiotas were assembled over shrimp postlarval to adult stages and a disease progression. The gut eukaryotic communities changed markedly as healthy shrimp aged, and converged toward an adult-microbiota configuration. However, the adult-like stability was distorted by disease exacerbation. A null model untangled that the deterministic processes that governed the gut eukaryotic assembly tended to be more important over healthy shrimp development, whereas this trend was inverted as the disease progressed. After ruling out the baseline of gut eukaryotes over shrimp ages, we identified disease-discriminatory taxa (species level afforded the highest accuracy of prediction) that characteristic of shrimp health status. The profiles of these taxa contributed an overall 92.4% accuracy in predicting shrimp health status. Notably, this model can accurately diagnose the onset of shrimp disease. Interspecies interaction analysis depicted how the disease-discriminatory taxa interacted with one another in sustaining shrimp health. Taken together, our findings offer novel insights into the underlying ecological processes that govern the assembly of gut eukaryotes over shrimp postlarval to adult stages and a disease progression. Intriguingly, the established model can quantitatively and accurately predict the incidences of shrimp disease.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

Science.gov (United States)

Schwager, Evelyn E; Sharma, Prashant P; Clarke, Thomas; Leite, Daniel J; Wierschin, Torsten; Pechmann, Matthias; Akiyama-Oda, Yasuko; Esposito, Lauren; Bechsgaard, Jesper; Bilde, Trine; Buffry, Alexandra D; Chao, Hsu; Dinh, Huyen; Doddapaneni, HarshaVardhan; Dugan, Shannon; Eibner, Cornelius; Extavour, Cassandra G; Funch, Peter; Garb, Jessica; Gonzalez, Luis B; Gonzalez, Vanessa L; Griffiths-Jones, Sam; Han, Yi; Hayashi, Cheryl; Hilbrant, Maarten; Hughes, Daniel S T; Janssen, Ralf; Lee, Sandra L; Maeso, Ignacio; Murali, Shwetha C; Muzny, Donna M; Nunes da Fonseca, Rodrigo; Paese, Christian L B; Qu, Jiaxin; Ronshaugen, Matthew; Schomburg, Christoph; Schönauer, Anna; Stollewerk, Angelika; Torres-Oliva, Montserrat; Turetzek, Natascha; Vanthournout, Bram; Werren, John H; Wolff, Carsten; Worley, Kim C; Bucher, Gregor; Gibbs, Richard A; Coddington, Jonathan; Oda, Hiroki; Stanke, Mario; Ayoub, Nadia A; Prpic, Nikola-Michael; Flot, Jean-François; Posnien, Nico; Richards, Stephen; McGregor, Alistair P

2017-07-31

The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

Mechanisms and regulation of DNA replication initiation in eukaryotes.

Science.gov (United States)

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

Science.gov (United States)

Castro-Carrera, T; Toral, P G; Frutos, P; McEwan, N R; Hervás, G; Abecia, L; Pinloche, E; Girdwood, S E; Belenguer, A

2014-03-01

Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition

Microbial diversity and dynamics throughout manufacturing and ripening of surface ripened semi-hard Danish Danbo cheeses investigated by culture-independent techniques

DEFF Research Database (Denmark)

Ryssel, Mia; Johansen, Pernille; Abu Al-Soud, Waleed

2015-01-01

) and pyrosequencing, using amplicons of 16S and 26S rRNA genes for prokaryotes and eukaryotes, respectively. With minor exceptions, the results from the culture-independent analyses correlated to the culture-dependent plating results. Even though the predominant microorganisms detected with the two culture...

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2012-01-01

in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis.

Science.gov (United States)

Smith, Alias J; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G; Jonsson, Zophonias O; Wohlschlegel, James A; Johnson, Patricia J

2011-04-01

A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ∼75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5' untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound specifically by a unique protein with a Myb-like DNA binding domain. The M5 element (CCTTT) overlaps the transcription start site and replaces the Inr as an alternative, gene-specific initiator element. Transcription specifically initiates at the second cytosine within M5, in contrast to characteristic initiation by RNA polymerase II at an adenosine. In promoters that combine M3 with either M5 or Inr, transcription initiation is regulated by the M3 motif.

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis▿

Science.gov (United States)

Smith, Alias J.; Chudnovsky, Lorissa; Simoes-Barbosa, Augusto; Delgadillo-Correa, Maria G.; Jonsson, Zophonias O.; Wohlschlegel, James A.; Johnson, Patricia J.