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Sample records for plastid rpotp rna

  1. Mediated Plastid RNA Editing in Plant Immunity

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    García-Andrade, Javier; Ramírez, Vicente; López, Ana; Vera, Pablo

    2013-01-01

    Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes. PMID:24204264

  2. Opposite roles of the Arabidopsis cytokinin receptors AHK2 and AHK3 in the expression of plastid genes and genes for the plastid transcriptional machinery during senescence.

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    Danilova, Maria N; Kudryakova, Natalia V; Doroshenko, Anastasia S; Zabrodin, Dmitry A; Rakhmankulova, Zulfira F; Oelmüller, Ralf; Kusnetsov, Victor V

    2017-03-01

    Cytokinin membrane receptors of the Arabidopsis thaliana AHK2 and AHK3 play opposite roles in the expression of plastid genes and genes for the plastid transcriptional machinery during leaf senescence Loss-of-function mutants of Arabidopsis thaliana were used to study the role of cytokinin receptors in the expression of chloroplast genes during leaf senescence. Accumulation of transcripts of several plastid-encoded genes is dependent on the АНК2/АНК3 receptor combination. АНК2 is particularly important at the final stage of plant development and, unlike АНК3, a positive regulator of leaf senescence. Cytokinin-dependent up-regulation of the nuclear encoded genes for chloroplast RNA polymerases RPOTp and RPOTmp suggests that the hormone controls plastid gene expression, at least in part, via the expression of nuclear genes for the plastid transcription machinery. This is further supported by cytokinin dependent regulation of genes for the nuclear encoded plastid σ-factors, SIG1-6, which code for components of the transcriptional apparatus in chloroplasts.

  3. Divergence of RNA polymerase ? subunits in angiosperm plastid genomes is mediated by genomic rearrangement

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    Blazier, J. Chris; Ruhlman, Tracey A.; Weng, Mao-Lun; Rehman, Sumaiyah K.; Sabir, Jamal S. M.; Jansen, Robert K.

    2016-01-01

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP ? subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled an...

  4. RNase P RNA from the Recently Evolved Plastid of Paulinella and from Algae

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    Pilar Bernal-Bayard

    2014-11-01

    Full Text Available The RNase P RNA catalytic subunit (RPR encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro.

  5. Divergence of RNA polymerase α subunits in angiosperm plastid genomes is mediated by genomic rearrangement.

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    Blazier, J Chris; Ruhlman, Tracey A; Weng, Mao-Lun; Rehman, Sumaiyah K; Sabir, Jamal S M; Jansen, Robert K

    2016-04-18

    Genes for the plastid-encoded RNA polymerase (PEP) persist in the plastid genomes of all photosynthetic angiosperms. However, three unrelated lineages (Annonaceae, Passifloraceae and Geraniaceae) have been identified with unusually divergent open reading frames (ORFs) in the conserved region of rpoA, the gene encoding the PEP α subunit. We used sequence-based approaches to evaluate whether these genes retain function. Both gene sequences and complete plastid genome sequences were assembled and analyzed from each of the three angiosperm families. Multiple lines of evidence indicated that the rpoA sequences are likely functional despite retaining as low as 30% nucleotide sequence identity with rpoA genes from outgroups in the same angiosperm order. The ratio of non-synonymous to synonymous substitutions indicated that these genes are under purifying selection, and bioinformatic prediction of conserved domains indicated that functional domains are preserved. One of the lineages (Pelargonium, Geraniaceae) contains species with multiple rpoA-like ORFs that show evidence of ongoing inter-paralog gene conversion. The plastid genomes containing these divergent rpoA genes have experienced extensive structural rearrangement, including large expansions of the inverted repeat. We propose that illegitimate recombination, not positive selection, has driven the divergence of rpoA.

  6. Constitutive Transcription and Stable RNA Accumulation in Plastids during the Conversion of Chloroplasts to Chromoplasts in Ripening Tomato Fruits 1

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    Marano, María Rosa; Carrillo, Néstor

    1992-01-01

    The size distribution of plastid transcripts during chromoplast differentiation in ripening tomato (Lycopersicon esculentum L.) fruits was determined using northern blot analysis. Hybridization of total cellular RNA from leaves and fruits with several tobacco chloroplast DNA probes showed distinct transcript patterns in chloroplasts and chromoplasts. We also compared transcriptional rates by probing immobilized DNA fragments of small size (representing about 85% of the plastid genome) with run-on transcripts from tomato plastids. The relative rates of transcription of the various DNA regions were very similar in chloro- and chromoplasts. Parallel determination of the steady-state levels of plastid RNA showed no strict correlation between synthesis rate and RNA accumulation. Differences in the relative abundance of transcripts between chloro- and chromoplasts were not very pronounced and were limited to a small number of genes. The results indicate that the conversion of chloroplasts to chromoplasts at the onset of tomato fruit ripening proceeds with no important variations in the relative transcription rates and with only moderate changes in the relative stability of plastid-encoded transcripts. Images Figure 1 Figure 4 PMID:16653091

  7. Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

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    Schuster, W; Brennicke, A

    1987-01-01

    We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

  8. Light-dependent, plastome-wide association of the plastid-encoded RNA polymerase with chloroplast DNA.

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    Finster, Sabrina; Eggert, Erik; Zoschke, Reimo; Weihe, Andreas; Schmitz-Linneweber, Christian

    2013-12-01

    Plastid genes are transcribed by two types of RNA polymerases: a plastid-encoded eubacterial-type RNA polymerase (PEP) and nuclear-encoded phage-type RNA polymerases (NEPs). To investigate the spatio-temporal expression of PEP, we tagged its α-subunit with a hemagglutinin epitope (HA). Transplastomic tobacco plants were generated and analyzed for the distribution of the tagged polymerase in plastid sub-fractions, and associated genes were identified under various light conditions. RpoA:HA was detected as early as the 3rd day after imbibition, and was constitutively expressed in green tissue over 60 days of plant development. We found that the tagged polymerase subunit preferentially associated with the plastid membranes, and was less abundant in the soluble stroma fraction. Attachment of RpoA:HA to the membrane fraction during early seedling development was independent of DNA, but at later stages of development, DNA appears to facilitate attachment of the polymerase to membranes. To survey PEP-dependent transcription units, we probed for nucleic acids enriched in RpoA:HA precipitates using a tobacco chloroplast whole-genome tiling array. The most strongly co-enriched DNA fragments represent photosynthesis genes (e.g. psbA, psbC, psbD and rbcL), whose expression is known to be driven by PEP promoters, while NEP-dependent genes were less abundant in RpoA:HA precipitates. Additionally, we demonstrate that the association of PEP with photosynthesis-related genes was reduced during the dark period, indicating that plastome-wide PEP-DNA association is a light-dependent process. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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

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    Xiao Li Shi

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

  10. Efficient Plastid Transformation in Arabidopsis.

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    Yu, Qiguo; Lutz, Kerry Ann; Maliga, Pal

    2017-09-01

    Plastid transformation is routine in tobacco ( Nicotiana tabacum ) but 100-fold less frequent in Arabidopsis ( Arabidopsis thaliana ), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2 , encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded ACC2 -defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance ( aadA ) gene and gfp , encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

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    Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

    2017-01-01

    Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

  12. Plastids and Carotenoid Accumulation.

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    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants.

  13. Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

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    Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

    2016-06-27

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

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    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  15. The plastid genome of the red macroalga Grateloupia taiwanensis (Halymeniaceae.

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    Michael S DePriest

    Full Text Available The complete plastid genome sequence of the red macroalga Grateloupia taiwanensis S.-M.Lin & H.-Y.Liang (Halymeniaceae, Rhodophyta is presented here. Comprising 191,270 bp, the circular DNA contains 233 protein-coding genes and 29 tRNA sequences. In addition, several genes previously unknown to red algal plastids are present in the genome of G. taiwanensis. The plastid genomes from G. taiwanensis and another florideophyte, Gracilaria tenuistipitata var. liui, are very similar in sequence and share significant synteny. In contrast, less synteny is shared between G. taiwanensis and the plastid genome representatives of Bangiophyceae and Cyanidiophyceae. Nevertheless, the gene content of all six red algal plastid genomes here studied is highly conserved, and a large core repertoire of plastid genes can be discerned in Rhodophyta.

  16. The Plastid Genome of the Red Macroalga Grateloupia taiwanensis (Halymeniaceae)

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    DePriest, Michael S.; Bhattacharya, Debashish; López-Bautista, Juan M.

    2013-01-01

    The complete plastid genome sequence of the red macroalga Grateloupia taiwanensis S.-M.Lin & H.-Y.Liang (Halymeniaceae, Rhodophyta) is presented here. Comprising 191,270 bp, the circular DNA contains 233 protein-coding genes and 29 tRNA sequences. In addition, several genes previously unknown to red algal plastids are present in the genome of G. taiwanensis. The plastid genomes from G. taiwanensis and another florideophyte, Gracilaria tenuistipitata var. liui, are very similar in sequence and share significant synteny. In contrast, less synteny is shared between G. taiwanensis and the plastid genome representatives of Bangiophyceae and Cyanidiophyceae. Nevertheless, the gene content of all six red algal plastid genomes here studied is highly conserved, and a large core repertoire of plastid genes can be discerned in Rhodophyta. PMID:23894297

  17. Evolution of red algal plastid genomes: ancient architectures, introns, horizontal gene transfer, and taxonomic utility of plastid markers.

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    Jan Janouškovec

    Full Text Available Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase - a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.

  18. Evolution of RLSB, a nuclear-encoded S1 domain RNA binding protein associated with post-transcriptional regulation of plastid-encoded rbcL mRNA in vascular plants.

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    Yerramsetty, Pradeep; Stata, Matt; Siford, Rebecca; Sage, Tammy L; Sage, Rowan F; Wong, Gane Ka-Shu; Albert, Victor A; Berry, James O

    2016-06-29

    RLSB, an S-1 domain RNA binding protein of Arabidopsis, selectively binds rbcL mRNA and co-localizes with Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within chloroplasts of C3 and C4 plants. Previous studies using both Arabidopsis (C3) and maize (C4) suggest RLSB homologs are post-transcriptional regulators of plastid-encoded rbcL mRNA. While RLSB accumulates in all Arabidopsis leaf chlorenchyma cells, in C4 leaves RLSB-like proteins accumulate only within Rubisco-containing bundle sheath chloroplasts of Kranz-type species, and only within central compartment chloroplasts in the single cell C4 plant Bienertia. Our recent evidence implicates this mRNA binding protein as a primary determinant of rbcL expression, cellular localization/compartmentalization, and photosynthetic function in all multicellular green plants. This study addresses the hypothesis that RLSB is a highly conserved Rubisco regulatory factor that occurs in the chloroplasts all higher plants. Phylogenetic analysis has identified RLSB orthologs and paralogs in all major plant groups, from ancient liverworts to recent angiosperms. RLSB homologs were also identified in algae of the division Charophyta, a lineage closely related to land plants. RLSB-like sequences were not identified in any other algae, suggesting that it may be specific to the evolutionary line leading to land plants. The RLSB family occurs in single copy across most angiosperms, although a few species with two copies were identified, seemingly randomly distributed throughout the various taxa, although perhaps correlating in some cases with known ancient whole genome duplications. Monocots of the order Poales (Poaceae and Cyperaceae) were found to contain two copies, designated here as RLSB-a and RLSB-b, with only RLSB-a implicated in the regulation of rbcL across the maize developmental gradient. Analysis of microsynteny in angiosperms revealed high levels of conservation across eudicot species and for both paralogs in

  19. Plastid genomes of two brown algae, Ectocarpus siliculosus and Fucus vesiculosus: further insights on the evolution of red-algal derived plastids

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    Corre Erwan

    2009-10-01

    Full Text Available Abstract Background Heterokont algae, together with cryptophytes, haptophytes and some alveolates, possess red-algal derived plastids. The chromalveolate hypothesis proposes that the red-algal derived plastids of all four groups have a monophyletic origin resulting from a single secondary endosymbiotic event. However, due to incongruence between nuclear and plastid phylogenies, this controversial hypothesis remains under debate. Large-scale genomic analyses have shown to be a powerful tool for phylogenetic reconstruction but insufficient sequence data have been available for red-algal derived plastid genomes. Results The chloroplast genomes of two brown algae, Ectocarpus siliculosus and Fucus vesiculosus, have been fully sequenced. These species represent two distinct orders of the Phaeophyceae, which is a major group within the heterokont lineage. The sizes of the circular plastid genomes are 139,954 and 124,986 base pairs, respectively, the size difference being due principally to the presence of longer inverted repeat and intergenic regions in E. siliculosus. Gene contents of the two plastids are similar with 139-148 protein-coding genes, 28-31 tRNA genes, and 3 ribosomal RNA genes. The two genomes also exhibit very similar rearrangements compared to other sequenced plastid genomes. The tRNA-Leu gene of E. siliculosus lacks an intron, in contrast to the F. vesiculosus and other heterokont plastid homologues, suggesting its recent loss in the Ectocarpales. Most of the brown algal plastid genes are shared with other red-algal derived plastid genomes, but a few are absent from raphidophyte or diatom plastid genomes. One of these regions is most similar to an apicomplexan nuclear sequence. The phylogenetic relationship between heterokonts, cryptophytes and haptophytes (collectively referred to as chromists plastids was investigated using several datasets of concatenated proteins from two cyanobacterial genomes and 18 plastid genomes, including

  20. Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

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    Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

    1990-01-01

    During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to ...

  1. Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein[W][OA

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    Kahlau, Sabine; Bock, Ralph

    2008-01-01

    Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts. PMID:18441214

  2. Does the mode of plastid inheritance influence plastid genome architecture?

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    Kate Crosby

    Full Text Available Plastid genomes show an impressive array of sizes and compactnesses, but the forces responsible for this variation are unknown. It has been argued that species with small effective genetic population sizes are less efficient at purging excess DNA from their genomes than those with large effective population sizes. If true, one may expect the primary mode of plastid inheritance to influence plastid DNA (ptDNA architecture. All else being equal, biparentally inherited ptDNAs should have a two-fold greater effective population size than those that are uniparentally inherited, and thus should also be more compact. Here, we explore the relationship between plastid inheritance pattern and ptDNA architecture, and consider the role of phylogeny in shaping our observations. Contrary to our expectations, we found no significant difference in plastid genome size or compactness between ptDNAs that are biparentally inherited relative to those that are uniparentally inherited. However, we also found that there was significant phylogenetic signal for the trait of mode of plastid inheritance. We also found that paternally inherited ptDNAs are significantly smaller (n = 19, p = 0.000001 than those that are maternally, uniparentally (when isogamous, or biparentally inherited. Potential explanations for this observation are discussed.

  3. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

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    Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  4. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

    KAUST Repository

    Gornik, Sebastian G.; Febrimarsa,; Cassin, Andrew M.; MacRae, James I.; Ramaprasad, Abhinay; Rchiad, ‍ Zineb; McConville, Malcolm J.; Bacic, Antony; McFadden, Geoffrey I.; Pain, Arnab; Waller, Ross F.

    2015-01-01

    Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes - notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium - highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite's host. Hematodinium sp. thus represents a further dimension of endosymbiosis-life after the organelle. © 2015, National Academy of Sciences. All rights reserved.

  5. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

    KAUST Repository

    Gornik, Sebastian G.

    2015-04-20

    Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes - notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium - highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite\\'s host. Hematodinium sp. thus represents a further dimension of endosymbiosis-life after the organelle. © 2015, National Academy of Sciences. All rights reserved.

  6. In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering

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    Yuyong Wu

    2017-08-01

    Full Text Available Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5′ untranslated region (UTR from gene10 of bacteriophage T7 and the transcript-stabilizing 3′UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was ~9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

  7. Quantitative proteomic analysis of intact plastids.

    Science.gov (United States)

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  8. Plastid and mitochondrial genomes of Coccophora langsdorfii (Fucales, Phaeophyceae and the utility of molecular markers.

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    Louis Graf

    Full Text Available Coccophora langsdorfii (Turner Greville (Fucales is an intertidal brown alga that is endemic to Northeast Asia and increasingly endangered by habitat loss and climate change. We sequenced the complete circular plastid and mitochondrial genomes of C. langsdorfii. The circular plastid genome is 124,450 bp and contains 139 protein-coding, 28 tRNA and 6 rRNA genes. The circular mitochondrial genome is 35,660 bp and contains 38 protein-coding, 25 tRNA and 3 rRNA genes. The structure and gene content of the C. langsdorfii plastid genome is similar to those of other species in the Fucales. The plastid genomes of brown algae in other orders share similar gene content but exhibit large structural recombination. The large in-frame insert in the cox2 gene in the mitochondrial genome of C. langsdorfii is typical of other brown algae. We explored the effect of this insertion on the structure and function of the cox2 protein. We estimated the usefulness of 135 plastid genes and 35 mitochondrial genes for developing molecular markers. This study shows that 29 organellar genes will prove efficient for resolving brown algal phylogeny. In addition, we propose a new molecular marker suitable for the study of intraspecific genetic diversity that should be tested in a large survey of populations of C. langsdorfii.

  9. Complete sequence and analysis of plastid genomes of two economically important red algae: Pyropia haitanensis and Pyropia yezoensis.

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    Li Wang

    Full Text Available Pyropia haitanensis and P. yezoensis are two economically important marine crops that are also considered to be research models to study the physiological ecology of intertidal seaweed communities, evolutionary biology of plastids, and the origins of sexual reproduction. This plastid genome information will facilitate study of breeding, population genetics and phylogenetics.We have fully sequenced using next-generation sequencing the circular plastid genomes of P. hatanensis (195,597 bp and P. yezoensis (191,975 bp, the largest of all the plastid genomes of the red lineage sequenced to date. Organization and gene contents of the two plastids were similar, with 211-213 protein-coding genes (including 29-31 unknown-function ORFs, 37 tRNA genes, and 6 ribosomal RNA genes, suggesting a largest coding capacity in the red lineage. In each genome, 14 protein genes overlapped and no interrupted genes were found, indicating a high degree of genomic condensation. Pyropia maintain an ancient gene content and conserved gene clusters in their plastid genomes, containing nearly complete repertoires of the plastid genes known in photosynthetic eukaryotes. Similarity analysis based on the whole plastid genome sequences showed the distance between P. haitanensis and P. yezoensis (0.146 was much smaller than that of Porphyra purpurea and P. haitanensis (0.250, and P. yezoensis (0.251; this supports re-grouping the two species in a resurrected genus Pyropia while maintaining P. purpurea in genus Porphyra. Phylogenetic analysis supports a sister relationship between Bangiophyceae and Florideophyceae, though precise phylogenetic relationships between multicellular red alage and chromists were not fully resolved.These results indicate that Pyropia have compact plastid genomes. Large coding capacity and long intergenic regions contribute to the size of the largest plastid genomes reported for the red lineage. Possessing the largest coding capacity and ancient gene

  10. Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii.

    Science.gov (United States)

    Funk, Helena T; Berg, Sabine; Krupinska, Karin; Maier, Uwe G; Krause, Kirsten

    2007-08-22

    The holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana. The newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing. Overall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression

  11. Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

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    Maier Uwe G

    2007-08-01

    Full Text Available Abstract Background The holoparasitic plant genus Cuscuta comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different Cuscuta species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species Cuscuta reflexa and Cuscuta gronovii. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of Epifagus virginiana, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in C. reflexa to a restricted photosynthetic activity and degenerated chloroplasts in C. gronovii to an achlorophyllous state in E. virginiana. Results The newly sequenced plastid genomes of C. reflexa and C. gronovii reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in C. gronovii and include (a the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c a significant reduction of RNA editing. Conclusion Overall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards

  12. Parallel loss of plastid introns and their maturase in the genus Cuscuta.

    Science.gov (United States)

    McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; Leebens-Mack, Jim; dePamphilis, Claude W

    2009-06-19

    Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

  13. Parallel loss of plastid introns and their maturase in the genus Cuscuta.

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    Joel R McNeal

    2009-06-01

    Full Text Available Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

  14. Plastid: nucleotide-resolution analysis of next-generation sequencing and genomics data.

    Science.gov (United States)

    Dunn, Joshua G; Weissman, Jonathan S

    2016-11-22

    Next-generation sequencing (NGS) informs many biological questions with unprecedented depth and nucleotide resolution. These assays have created a need for analytical tools that enable users to manipulate data nucleotide-by-nucleotide robustly and easily. Furthermore, because many NGS assays encode information jointly within multiple properties of read alignments - for example, in ribosome profiling, the locations of ribosomes are jointly encoded in alignment coordinates and length - analytical tools are often required to extract the biological meaning from the alignments before analysis. Many assay-specific pipelines exist for this purpose, but there remains a need for user-friendly, generalized, nucleotide-resolution tools that are not limited to specific experimental regimes or analytical workflows. Plastid is a Python library designed specifically for nucleotide-resolution analysis of genomics and NGS data. As such, Plastid is designed to extract assay-specific information from read alignments while retaining generality and extensibility to novel NGS assays. Plastid represents NGS and other biological data as arrays of values associated with genomic or transcriptomic positions, and contains configurable tools to convert data from a variety of sources to such arrays. Plastid also includes numerous tools to manipulate even discontinuous genomic features, such as spliced transcripts, with nucleotide precision. Plastid automatically handles conversion between genomic and feature-centric coordinates, accounting for splicing and strand, freeing users of burdensome accounting. Finally, Plastid's data models use consistent and familiar biological idioms, enabling even beginners to develop sophisticated analytical workflows with minimal effort. Plastid is a versatile toolkit that has been used to analyze data from multiple NGS assays, including RNA-seq, ribosome profiling, and DMS-seq. It forms the genomic engine of our ORF annotation tool, ORF-RATER, and is readily

  15. Coordinated rates of evolution between interacting plastid and nuclear genes in Geraniaceae.

    Science.gov (United States)

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal; Blazier, J Chris; Jansen, Robert K

    2015-03-01

    Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae. © 2015 American Society of Plant Biologists. All rights reserved.

  16. Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta.

    Science.gov (United States)

    McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; de Pamphilis, Claude W

    2007-10-24

    Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.

  17. Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta

    Directory of Open Access Journals (Sweden)

    Kuehl Jennifer V

    2007-10-01

    Full Text Available Abstract Background Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Results Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Conclusion Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.

  18. Phytochrome-dependent coordinate control of distinct aspects of nuclear and plastid gene expression during anterograde signalling and photomorphogenesis

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    Sookyung eOh

    2014-04-01

    Full Text Available Light perception by photoreceptors impacts plastid transcription, development, and differentiation. This photoreceptor-dependent activity suggests a mechanism for photoregulation of gene expression in the nucleus and plastid that serves to coordinate expression of critical genes of these two organelles. This coordinate expression is required for proper stoichiometric accumulation of components needed for assembly of plastids, photosynthetic light-harvesting complexes and components such as phytochromes. Chloroplast-targeted sigma factors, which function together with the plastid-encoded RNA polymerase to regulate expression of plastid-encoded genes, and nuclear-encoded plastid development factors, such as GLK1 and GLK2, are targets of phytochrome regulation. Such phytochrome-dependent functions are hypothesized to allow light-dependent regulation, and feasibly tuning, of plastid components and function in response to changes in the external environment, which directly affects photosynthesis and the potential for light-induced damage. When the size and protein composition of the light-harvesting complexes are not tuned to the external environment, imbalances in electron transport can impact the cellular redox state and cause cellular damage. We show that phytochromes specifically regulate the expression of multiple factors that function to modulate plastid transcription and, thus, provide a paradigm for coordinate expression of the nuclear and plastid genomes in response to changes in external light conditions. As phytochromes respond to changes in the prevalent wavelengths of light and light intensity, we propose that specific phytochrome-dependent molecular mechanisms are used during light-dependent signaling between the nucleus and chloroplast during photomorphogenesis to coordinate chloroplast development with plant developmental stage and the external environment.

  19. Ties that bind: the integration of plastid signalling pathways in plant cell metabolism.

    Science.gov (United States)

    Brunkard, Jacob O; Burch-Smith, Tessa M

    2018-04-13

    Plastids are critical organelles in plant cells that perform diverse functions and are central to many metabolic pathways. Beyond their major roles in primary metabolism, of which their role in photosynthesis is perhaps best known, plastids contribute to the biosynthesis of phytohormones and other secondary metabolites, store critical biomolecules, and sense a range of environmental stresses. Accordingly, plastid-derived signals coordinate a host of physiological and developmental processes, often by emitting signalling molecules that regulate the expression of nuclear genes. Several excellent recent reviews have provided broad perspectives on plastid signalling pathways. In this review, we will highlight recent advances in our understanding of chloroplast signalling pathways. Our discussion focuses on new discoveries illuminating how chloroplasts determine life and death decisions in cells and on studies elucidating tetrapyrrole biosynthesis signal transduction networks. We will also examine the role of a plastid RNA helicase, ISE2, in chloroplast signalling, and scrutinize intriguing results investigating the potential role of stromules in conducting signals from the chloroplast to other cellular locations. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  20. A Targeted Enrichment Strategy for Massively Parallel Sequencing of Angiosperm Plastid Genomes

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    Gregory W. Stull

    2013-02-01

    Full Text Available Premise of the study: We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms. Methods and Results: A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots, which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×, even for the two monocots. Conclusions: Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving 50× mean coverage. However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96 available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms.

  1. Plastid and Stromule Morphogenesis in Tomato

    Science.gov (United States)

    PYKE, KEVIN A.; HOWELLS, CAROLINE A.

    2002-01-01

    By using green fluorescent protein targeted to the plastid organelle in tomato (Lycopersicon esculentum Mill.), the morphology of plastids and their associated stromules in epidermal cells and trichomes from stems and petioles and in the chromoplasts of pericarp cells in the tomato fruit has been revealed. A novel characteristic of tomato stromules is the presence of extensive bead‐like structures along the stromules that are often observed as free vesicles, distinct from and apparently unconnected to the plastid body. Interconnections between the red pigmented chromoplast bodies are common in fruit pericarp cells suggesting that chromoplasts could form a complex network in this cell type. The potential implications for carotenoid biosynthesis in tomato fruit and for vesicles originating from beaded stromules as a secretory mechanism for plastids in glandular trichomes of tomato is discussed. PMID:12466096

  2. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  3. Plastids: the Green Frontiers for Vaccine Production

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    Mohammad Tahir eWaheed

    2015-11-01

    Full Text Available Infectious diseases pose an increasing risk to health, especially in developing countries. Vaccines are available to either cure or prevent many of these diseases. However, there are certain limitations related to these vaccines, mainly the costs, which make these vaccines mostly unaffordable for people in resource poor countries. These costs are mainly related to production and purification of the products manufactured from fermenter-based systems. Plastid biotechnology has become an attractive platform to produce biopharmaceuticals in large amounts and cost-effectively. This is mainly due to high copy number of plastids DNA in mature chloroplasts, a characteristic particularly important for vaccine production in large amounts. An additional advantage lies in the maternal inheritance of plastids in most plant species, which addresses the regulatory concerns related to transgenic plants. These and many other aspects of plastids will be discussed in the present review, especially those that particularly make these green biofactories an attractive platform for vaccine production. A summary of recent vaccine antigens against different human diseases expressed in plastids will also be presented.

  4. Stable plastid transformation in Scoparia dulcis L.

    Science.gov (United States)

    Muralikrishna, Narra; Srinivas, Kota; Kumar, Kalva Bharath; Sadanandam, Abbagani

    2016-10-01

    In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR / t rnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

  5. The plastid genomes of flowering plants.

    Science.gov (United States)

    Ruhlman, Tracey A; Jansen, Robert K

    2014-01-01

    The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering.

  6. Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

    Science.gov (United States)

    Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

    1990-01-01

    During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant. The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts. Images Fig. 1 Fig. 2 Fig. 3. Fig. 4. Fig. 5. PMID:2303026

  7. The myth of interconnected plastids and related phenomena.

    Science.gov (United States)

    Schattat, Martin H; Barton, Kiah A; Mathur, Jaideep

    2015-01-01

    Studies spread over nearly two and a half centuries have identified the primary plastid in autotrophic algae and plants as a pleomorphic, multifunctional organelle comprising of a double-membrane envelope enclosing an organization of internal membranes submerged in a watery stroma. All plastid units have been observed extending and retracting thin stroma-filled tubules named stromules sporadically. Observations on living plant cells often convey the impression that stromules connect two or more independent plastids with each other. When photo-bleaching techniques were used to suggest that macromolecules such as the green fluorescent protein could flow between already interconnected plastids, for many people this impression changed to conviction. However, it was noticed only recently that the concept of protein flow between plastids rests solely on the words "interconnected plastids" for which details have never been provided. We have critically reviewed botanical literature dating back to the 1880s for understanding this term and the phenomena that have become associated with it. We find that while meticulously detailed ontogenic studies spanning nearly 150 years have established the plastid as a singular unit organelle, there is no experimental support for the idea that interconnected plastids exist under normal conditions of growth and development. In this review, while we consider several possibilities that might allow a single elongated plastid to be misinterpreted as two or more interconnected plastids, our final conclusion is that the concept of direct protein flow between plastids is based on an unfounded assumption.

  8. Plastome-Genome Interactions Affect Plastid Transmission in Oenothera

    Science.gov (United States)

    Chiu, W. L.; Sears, B. B.

    1993-01-01

    Plastids of Oenothera, the evening primrose, can be transmitted to the progeny from both parents. In a constant nuclear background, the frequency of biparental plastid transmission is determined by the types of plastid genomes (plastomes) involved in the crosses. In this study, the impact of nuclear genomes on plastid inheritance was analyzed. In general, the transmission efficiency of each plastome correlated strongly with its compatibility with the nuclear genome of the progeny, suggesting that plastome-genome interactions can influence plastid transmission by affecting the efficiency of plastid multiplication after fertilization. Lower frequencies of plastid transmission from the paternal side were observed when the pollen had poor vigor due to an incompatible plastome-genome combination, indicating that plastome-genome interactions may also affect the input of plastids at fertilization. Parental traits that affect the process of fertilization can also have an impact on plastid transmission. Crosses using maternal parents with long styles or pollen with relatively low growth capacity resulted in reduced frequencies of paternal plastid transmission. These observations suggest that degeneration of pollen plastids may occur as the time interval between pollination and fertilization is lengthened. PMID:8462856

  9. Why does biparental plastid inheritance revive in angiosperms?

    Science.gov (United States)

    Zhang, Quan; Sodmergen

    2010-03-01

    It is widely believed that plastid and mitochondrial genomes are inherited through the maternal parent. In plants, however, paternal transmission of these genomes is frequently observed, especially for the plastid genome. A male gametic trait, called potential biparental plastid inheritance (PBPI), occurs in up to 20% of angiosperm genera, implying a strong tendency for plastid transmission from the male lineage. Why do plants receive organelles from the male parents? Are there clues in plastids that will help to elucidate the evolution of plants? Reconstruction of the ancestral state of plastid inheritance patterns in a phylogenetic context provides insights into these questions. In particular, a recent report demonstrated the unilateral occurrence of PBPI in angiosperms. This result implies that nuclear cytoplasmic conflicts, a basic driving force for altering the mode of organelle inheritance, might have arisen specifically in angiosperms. Based on existing evidence, it is likely that biparental inheritance may have occurred to rescue angiosperm species with defective plastids.

  10. Plastid Proteomic Analysis in Tomato Fruit Development.

    Directory of Open Access Journals (Sweden)

    Miho Suzuki

    Full Text Available To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein and HrBP1 (harpin binding protein-1 in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

  11. Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms.

    Science.gov (United States)

    Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

    2006-08-31

    Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats > or = 30 bp with a sequence identity > or = 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These

  12. Complete plastid genome sequence of Daucus carota: Implications for biotechnology and phylogeny of angiosperms

    Directory of Open Access Journals (Sweden)

    Ruhlman Tracey

    2006-08-01

    Full Text Available Abstract Background Carrot (Daucus carota is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. Results The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats ≥ 30 bp with a sequence identity ≥ 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP and maximum likelihood (ML were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. Conclusion The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap for the sister relationship of

  13. Carotenoid Metabolism in Plants: The Role of Plastids.

    Science.gov (United States)

    Sun, Tianhu; Yuan, Hui; Cao, Hongbo; Yazdani, Mohammad; Tadmor, Yaakov; Li, Li

    2018-01-08

    Carotenoids are indispensable to plants and critical in human diets. Plastids are the organelles for carotenoid biosynthesis and storage in plant cells. They exist in various types, which include proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. These plastids have dramatic differences in their capacity to synthesize and sequester carotenoids. Clearly, plastids play a central role in governing carotenogenic activity, carotenoid stability, and pigment diversity. Understanding of carotenoid metabolism and accumulation in various plastids expands our view on the multifaceted regulation of carotenogenesis and facilitates our efforts toward developing nutrient-enriched food crops. In this review, we provide a comprehensive overview of the impact of various types of plastids on carotenoid biosynthesis and accumulation, and discuss recent advances in our understanding of the regulatory control of carotenogenesis and metabolic engineering of carotenoids in light of plastid types in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  14. Plastid transformation in potato: Solanum tuberosum.

    Science.gov (United States)

    Valkov, Vladimir T; Gargano, Daniela; Scotti, Nunzia; Cardi, Teodoro

    2014-01-01

    Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts.

  15. Vesicles Are Persistent Features of Different Plastids.

    Science.gov (United States)

    Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

    2016-10-01

    Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Respiratory processes in non-photosynthetic plastids

    Science.gov (United States)

    Renato, Marta; Boronat, Albert; Azcón-Bieto, Joaquín

    2015-01-01

    Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(P)H to oxygen. This respiratory chain involves the NAD(P)H dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX), and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids. PMID:26236317

  17. Respiratory processes in non-photosynthetic plastids

    Directory of Open Access Journals (Sweden)

    Marta eRenato

    2015-07-01

    Full Text Available Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(PH to oxygen. This respiratory chain involves the NAD(PH dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX, and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids.

  18. On being the right size as an animal with plastids

    NARCIS (Netherlands)

    C. Rauch (Cessa); P. Jahns (Peter); A.G.M. Tielens (Aloysius); D.B. Gould (Douglas ); W.F. Martin (William F.)

    2017-01-01

    textabstractPlastids typically reside in plant or algal cells—with one notable exception. There is one group of multicellular animals, sea slugs in the order Sacoglossa, members of which feed on siphonaceous algae. The slugs sequester the ingested plastids in the cytosol of cells in their digestive

  19. On Being the Right Size as an Animal with Plastids

    NARCIS (Netherlands)

    Rauch, Cessa; Jahns, Peter; Tielens, Aloysius G M; Gould, Sven B; Martin, William F

    2017-01-01

    Plastids typically reside in plant or algal cells-with one notable exception. There is one group of multicellular animals, sea slugs in the order Sacoglossa, members of which feed on siphonaceous algae. The slugs sequester the ingested plastids in the cytosol of cells in their digestive gland,

  20. Plant cells without detectable plastids are generated in the crumpled leaf mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Yuling; Asano, Tomoya; Fujiwara, Makoto T; Yoshida, Shigeo; Machida, Yasunori; Yoshioka, Yasushi

    2009-05-01

    Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. It is unclear, however, whether cells without plastids are generated when plastid division is suppressed. The crumpled leaf (crl) mutant of Arabidopsis thaliana is a plastid division mutant that displays severe abnormalities in plastid division and plant development. We show that the crl mutant contains cells lacking detectable plastids; this situation probably results from an unequal partitioning of plastids to each daughter cell. Our results suggest that crl has a partial defect in plastid expansion, which is suggested to be important in the partitioning of plastids to daughter cells when plastid division is suppressed. The absence of cells without detectable plastids in the accumulation and replication of chloroplasts 6 (arc6) mutant, another plastid division mutant of A. thaliana having no significant defects in plant morphology, suggests that the generation of cells without detectable plastids is one of the causes of the developmental abnormalities seen in crl plants. We also demonstrate that plastids with trace or undetectable amounts of chlorophyll are generated from enlarged plastids by a non-binary fission mode of plastid replication in both crl and arc6.

  1. Fatty acid biosynthesis in pea root plastids

    International Nuclear Information System (INIS)

    Stahl, R.J.; Sparace, S.A.

    1989-01-01

    Fatty acid biosynthesis from [1- 14 C]acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 μM acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl 2 , 1 mM each of the MnCl 2 and glycerol-3-phosphate, 15 mM KHCO 3 , and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 μg/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO 3 , divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg 2+ and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor

  2. Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana.

    Science.gov (United States)

    Morikawa, Kazuya; Shiina, Takashi; Murakami, Shinya; Toyoshima, Yoshinori

    2002-03-13

    Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. Stable plastid transformation in lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Lelivelt, Cilia L C; McCabe, Matthew S; Newell, Christine A; Desnoo, C Bastiaan; van Dun, Kees M P; Birch-Machin, Ian; Gray, John C; Mills, Kingston H G; Nugent, Jacqueline M

    2005-08-01

    Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T(1) plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.

  6. Rapid and accurate pyrosequencing of angiosperm plastid genomes

    Science.gov (United States)

    Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

    2006-01-01

    Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid

  7. Rapid and accurate pyrosequencing of angiosperm plastid genomes

    Directory of Open Access Journals (Sweden)

    Farmerie William G

    2006-08-01

    Full Text Available Abstract Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20 System (454 Life Sciences Corporation, to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae and Platanus occidentalis (Platanaceae. Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy

  8. Non-photosynthetic plastids as hosts for metabolic engineering.

    Science.gov (United States)

    Mellor, Silas Busck; Behrendorff, James B Y H; Nielsen, Agnieszka Zygadlo; Jensen, Poul Erik; Pribil, Mathias

    2018-04-13

    Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource. Plant cells contain organelles called plastids that retain their own genome, harbour unique biosynthetic pathways and differentiate into distinct plastid types upon environmental and developmental cues. Chloroplasts, the plastid type hosting the photosynthetic processes in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis and storage of particular classes of compounds, might prove more suitable for engineering the production and storage of non-native metabolites without affecting plant fitness. This review provides the current state of knowledge on the molecular mechanisms involved in plastid differentiation and focuses on non-photosynthetic plastids as alternative biotechnological platforms for metabolic engineering. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  9. The plastid genome of Eutreptiella provides a window into the process of secondary endosymbiosis of plastid in euglenids.

    Directory of Open Access Journals (Sweden)

    Štěpánka Hrdá

    Full Text Available Euglenids are a group of protists that comprises species with diverse feeding modes. One distinct and diversified clade of euglenids is photoautotrophic, and its members bear green secondary plastids. In this paper we present the plastid genome of the euglenid Eutreptiella, which we assembled from 454 sequencing of Eutreptiella gDNA. Comparison of this genome and the only other available plastid genomes of photosynthetic euglenid, Euglena gracilis, revealed that they contain a virtually identical set of 57 protein coding genes, 24 genes fewer than the genome of Pyramimonas parkeae, the closest extant algal relative of the euglenid plastid. Searching within the transcriptomes of Euglena and Eutreptiella showed that 6 of the missing genes were transferred to the nucleus of the euglenid host while 18 have been probably lost completely. Euglena and Eutreptiella represent the deepest bifurcation in the photosynthetic clade, and therefore all these gene transfers and losses must have happened before the last common ancestor of all known photosynthetic euglenids. After the split of Euglena and Eutreptiella only one additional gene loss took place. The conservation of gene content in the two lineages of euglenids is in contrast to the variability of gene order and intron counts, which diversified dramatically. Our results show that the early secondary plastid of euglenids was much more susceptible to gene losses and endosymbiotic gene transfers than the established plastid, which is surprisingly resistant to changes in gene content.

  10. Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae.

    Science.gov (United States)

    Szczecińska, Monika; Sawicki, Jakub

    2015-09-15

    The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161-162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. The determination of complete

  11. Plastid and mitochondrion genomic sequences from Arctic Chlorella sp. ArM0029B

    Science.gov (United States)

    2014-01-01

    Background Chorella is the representative taxon of Chlorellales in Trebouxiophyceae, and its chloroplast (cp) genomic information has been thought to depend only on studies concerning Chlorella vulgaris and GenBank information of C. variablis. Mitochondrial (mt) genomic information regarding Chlorella is currently unavailable. To elucidate the evolution of organelle genomes and genetic information of Chlorella, we have sequenced and characterized the cp and mt genomes of Arctic Chlorella sp. ArM0029B. Results The 119,989-bp cp genome lacking inverted repeats and 65,049-bp mt genome were sequenced. The ArM0029B cp genome contains 114 conserved genes, including 32 tRNA genes, 3 rRNA genes, and 79 genes encoding proteins. Chlorella cp genomes are highly rearranged except for a Chlorella-specific six-gene cluster, and the ArM0029B plastid resembles that of Chlorella variabilis except for a 15-kb gene cluster inversion. In the mt genome, 62 conserved genes, including 27 tRNA genes, 3 rRNA genes, and 32 genes encoding proteins were determined. The mt genome of ArM0029B is similar to that of the non-photosynthetic species Prototheca and Heicosporidium. The ArM0029B mt genome contains a group I intron, with an ORF containing two LAGLIDADG motifs, in cox1. The intronic ORF is shared by C. vulgaris and Prototheca. The phylogeny of the plastid genome reveals that ArM0029B showed a close relationship of Chlorella to Parachlorella and Oocystis within Chlorellales. The distribution of the cox1 intron at 721 support membership in the order Chlorellales. Mitochondrial phylogenomic analyses, however, indicated that ArM0029B shows a greater affinity to MX-AZ01 and Coccomyxa than to the Helicosporidium-Prototheca clade, although the detailed phylogenetic relationships among the three taxa remain to be resolved. Conclusions The plastid genome of ArM0029B is similar to that of C. variabilis. The mt sequence of ArM0029B is the first genome to be reported for Chlorella. Chloroplast

  12. Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

    Science.gov (United States)

    Fu, Xiumin; Kong, Wenbin; Peng, Gang; Zhou, Jingyi; Azam, Muhammad; Xu, Changjie; Grierson, Don; Chen, Kunsong

    2012-01-01

    Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g−1 and 13 μg g−1 fresh weight (FW), respectively, and for BS 19 μg g−1 and 0.27 μg g−1 FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit. PMID:21994170

  13. Plastid-like Seq in mt Genome - RMG | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available deletions in the plastid DNA sequence (Number of deletion sites is shown in parentheses) Insertion Number o...f nucleotide insertions in the plastid DNA sequence (Number of insertion sites is shown in parentheses) Homo

  14. Intragenomic spread of plastid-targeting presequences in the coccolithophore Emiliania huxleyi.

    Science.gov (United States)

    Burki, Fabien; Hirakawa, Yoshihisa; Keeling, Patrick J

    2012-09-01

    Nucleus-encoded plastid-targeted proteins of photosynthetic organisms are generally equipped with an N-terminal presequence required for crossing the plastid membranes. The acquisition of these presequences played a fundamental role in the establishment of plastids. Here, we report a unique case of two non-homologous proteins possessing completely identical presequences consisting of a bipartite plastid-targeting signal in the coccolithophore Emiliania huxleyi. We further show that this presequence is highly conserved in five additional proteins that did not originally function in plastids, representing de novo plastid acquisitions. These are among the most recent cases of presequence spreading from gene to gene and shed light on important evolutionary processes that have been usually erased by the ancient history of plastid evolution. We propose a mechanism of acquisition involving genomic duplications and gene replacement through non-homologous recombination that may have played a more general role for equipping proteins with targeting information.

  15. Gene expression in isolated plastids from fruits of capsicum annum

    International Nuclear Information System (INIS)

    Powell, D.S.; Pryke, J.A.

    1987-01-01

    Plastids were obtained from the ripening fruits of Capsicum annum, and incubated in vitro in the presence of [ 35 S]methionine(Met). There was polypeptide synthesis at all stages of pepper tissue studied in both chloroplasts and chromoplasts, dependent on the addition of nuclioside triphosphates and phosphoenolpyruvate and inhibited by D-threo-chloramphenicol. l8. refs. (author)

  16. Production Of Cellulase In Plastids Of Transgenic Plants

    Science.gov (United States)

    Lamppa, Gayle

    2002-08-06

    A genetic construct encoding a fusion protein including endogluconase E1 and a transit peptide is used to transform plants. The plants produce cellulase by expressing the genetic construct. The cellulase is targeted to plastids and can be collected and purified.

  17. Comparative analyses of plastid genomes from fourteen Cornales species: inferences for phylogenetic relationships and genome evolution.

    Science.gov (United States)

    Fu, Chao-Nan; Li, Hong-Tao; Milne, Richard; Zhang, Ting; Ma, Peng-Fei; Yang, Jing; Li, De-Zhu; Gao, Lian-Ming

    2017-12-08

    The Cornales is the basal lineage of the asterids, the largest angiosperm clade. Phylogenetic relationships within the order were previously not fully resolved. Fifteen plastid genomes representing 14 species, ten genera and seven families of Cornales were newly sequenced for comparative analyses of genome features, evolution, and phylogenomics based on different partitioning schemes and filtering strategies. All plastomes of the 14 Cornales species had the typical quadripartite structure with a genome size ranging from 156,567 bp to 158,715 bp, which included two inverted repeats (25,859-26,451 bp) separated by a large single-copy region (86,089-87,835 bp) and a small single-copy region (18,250-18,856 bp) region. These plastomes encoded the same set of 114 unique genes including 31 transfer RNA, 4 ribosomal RNA and 79 coding genes, with an identical gene order across all examined Cornales species. Two genes (rpl22 and ycf15) contained premature stop codons in seven and five species respectively. The phylogenetic relationships among all sampled species were fully resolved with maximum support. Different filtering strategies (none, light and strict) of sequence alignment did not have an effect on these relationships. The topology recovered from coding and noncoding data sets was the same as for the whole plastome, regardless of filtering strategy. Moreover, mutational hotspots and highly informative regions were identified. Phylogenetic relationships among families and intergeneric relationships within family of Cornales were well resolved. Different filtering strategies and partitioning schemes do not influence the relationships. Plastid genomes have great potential to resolve deep phylogenetic relationships of plants.

  18. Instability of plastid DNA in the nuclear genome.

    Directory of Open Access Journals (Sweden)

    Anna E Sheppard

    2009-01-01

    Full Text Available Functional gene transfer from the plastid (chloroplast and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.

  19. The plastid genome of Eutreptiella provides a window into the process of secondary endosymbiosis of plastid in euglenids

    Czech Academy of Sciences Publication Activity Database

    Hrdá, Š.; Fousek, Jan; Szabová, J.; Vlček, Čestmír; Hampl, V.

    2012-01-01

    Roč. 7, č. 3 (2012), e33746 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP506/11/1320 Institutional support: RVO:68378050 Keywords : euglenid plastid * Eutreptiella * phylogeny Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

  20. The complete plastome of macaw palm [Acrocomia aculeata (Jacq.) Lodd. ex Mart.] and extensive molecular analyses of the evolution of plastid genes in Arecaceae.

    Science.gov (United States)

    de Santana Lopes, Amanda; Gomes Pacheco, Túlio; Nimz, Tabea; do Nascimento Vieira, Leila; Guerra, Miguel P; Nodari, Rubens O; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Rogalski, Marcelo

    2018-04-01

    The plastome of macaw palm was sequenced allowing analyses of evolution and molecular markers. Additionally, we demonstrated that more than half of plastid protein-coding genes in Arecaceae underwent positive selection. Macaw palm is a native species from tropical and subtropical Americas. It shows high production of oil per hectare reaching up to 70% of oil content in fruits and an interesting plasticity to grow in different ecosystems. Its domestication and breeding are still in the beginning, which makes the development of molecular markers essential to assess natural populations and germplasm collections. Therefore, we sequenced and characterized in detail the plastome of macaw palm. A total of 221 SSR loci were identified in the plastome of macaw palm. Additionally, eight polymorphism hotspots were characterized at level of subfamily and tribe. Moreover, several events of gain and loss of RNA editing sites were found within the subfamily Arecoideae. Aiming to uncover evolutionary events in Arecaceae, we also analyzed extensively the evolution of plastid genes. The analyses show that highly divergent genes seem to evolve in a species-specific manner, suggesting that gene degeneration events may be occurring within Arecaceae at the level of genus or species. Unexpectedly, we found that more than half of plastid protein-coding genes are under positive selection, including genes for photosynthesis, gene expression machinery and other essential plastid functions. Furthermore, we performed a phylogenomic analysis using whole plastomes of 40 taxa, representing all subfamilies of Arecaceae, which placed the macaw palm within the tribe Cocoseae. Finally, the data showed here are important for genetic studies in macaw palm and provide new insights into the evolution of plastid genes and environmental adaptation in Arecaceae.

  1. Mutations in a plastid-localized elongation factor G alter early stages of plastid development in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hangarter Roger P

    2007-07-01

    Full Text Available Abstract Background Proper development of plastids in embryo and seedling tissues is critical for plant development. During germination, plastids develop to perform many critical functions that are necessary to establish the seedling for further growth. A growing body of work has demonstrated that components of the plastid transcription and translation machinery must be present and functional to establish the organelle upon germination. Results We have identified Arabidopsis thaliana mutants in a gene that encodes a plastid-targeted elongation factor G (SCO1 that is essential for plastid development during embryogenesis since two T-DNA insertion mutations in the coding sequence (sco1-2 and sco1-3 result in an embryo-lethal phenotype. In addition, a point mutation allele (sco1-1 and an allele with a T-DNA insertion in the promoter (sco1-4 of SCO1 display conditional seedling-lethal phenotypes. Seedlings of these alleles exhibit cotyledon and hypocotyl albinism due to improper chloroplast development, and normally die shortly after germination. However, when germinated on media supplemented with sucrose, the mutant plants can produce photosynthetically-active green leaves from the apical meristem. Conclusion The developmental stage-specific phenotype of the conditional-lethal sco1 alleles reveals differences in chloroplast formation during seedling germination compared to chloroplast differentiation in cells derived from the shoot apical meristem. Our identification of embryo-lethal mutant alleles in the Arabidopsis elongation factor G indicates that SCO1 is essential for plant growth, consistent with its predicted role in chloroplast protein translation.

  2. Entire plastid phylogeny of the carrot genus (Daucus, Apiaceae): Concordance with nuclear data and mitochondrial and nuclear DNA insertions to the plastid.

    Science.gov (United States)

    Spooner, David M; Ruess, Holly; Iorizzo, Massimo; Senalik, Douglas; Simon, Philipp

    2017-02-01

    We explored the phylogenetic utility of entire plastid DNA sequences in Daucus and compared the results with prior phylogenetic results using plastid and nuclear DNA sequences. We used Illumina sequencing to obtain full plastid sequences of 37 accessions of 20 Daucus taxa and outgroups, analyzed the data with phylogenetic methods, and examined evidence for mitochondrial DNA transfer to the plastid ( Dc MP). Our phylogenetic trees of the entire data set were highly resolved, with 100% bootstrap support for most of the external and many of the internal clades, except for the clade of D. carota and its most closely related species D. syrticus . Subsets of the data, including regions traditionally used as phylogenetically informative regions, provide various degrees of soft congruence with the entire data set. There are areas of hard incongruence, however, with phylogenies using nuclear data. We extended knowledge of a mitochondrial to plastid DNA insertion sequence previously named Dc MP and identified the first instance in flowering plants of a sequence of potential nuclear genome origin inserted into the plastid genome. There is a relationship of inverted repeat junction classes and repeat DNA to phylogeny, but no such relationship with nonsynonymous mutations. Our data have allowed us to (1) produce a well-resolved plastid phylogeny of Daucus , (2) evaluate subsets of the entire plastid data for phylogeny, (3) examine evidence for plastid and nuclear DNA phylogenetic incongruence, and (4) examine mitochondrial and nuclear DNA insertion into the plastid. © 2017 Spooner et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons public domain license (CC0 1.0).

  3. CyanoClust: comparative genome resources of cyanobacteria and plastids.

    Science.gov (United States)

    Sasaki, Naobumi V; Sato, Naoki

    2010-01-01

    Cyanobacteria, which perform oxygen-evolving photosynthesis as do chloroplasts of plants and algae, are one of the best-studied prokaryotic phyla and one from which many representative genomes have been sequenced. Lack of a suitable comparative genomic database has been a problem in cyanobacterial genomics because many proteins involved in physiological functions such as photosynthesis and nitrogen fixation are not catalogued in commonly used databases, such as Clusters of Orthologous Proteins (COG). CyanoClust is a database of homolog groups in cyanobacteria and plastids that are produced by the program Gclust. We have developed a web-server system for the protein homology database featuring cyanobacteria and plastids. Database URL: http://cyanoclust.c.u-tokyo.ac.jp/.

  4. Demonstration of paternal inheritance of plastids in Picea (Pinaceae)

    International Nuclear Information System (INIS)

    Stine, M.

    1988-01-01

    Chloroplast DNA (cpDNA) was purified from Picea glauca, P. pungens, P. engelmannii, and P. omorika, and was digested with several restriction endonucleases. Interspecific restriction fragment length polymorphisms (RFLPs) of cpDNA were identified. The RFLPs were identified as cpDNA by the hybridization of cloned, 32 -P labeled, petunia cpDNA to the polymorphic bands, and by the lack of hybridization of a cloned and labeled mtDNA probe from maize. Chloroplast DNA RFLPs that showed no intraspecific variation when examined across the natural range for each species, were used as markers to follow the inheritance of plastids in interspecific hybrids. The inheritance of plastids was determined for F 1 -hybrids from reciprocal crosses of P. glauca and P. pungens, P. glauca and P. omorika, and F 1 -hybrids of P. engelmannii x pungens. All 31 F 1 -hybrids examined showed the cpDNA genotypes of the pollen parent, or the paternal species

  5. Cuscuta europaea plastid apparatus in various developmental stages

    Science.gov (United States)

    Švubová, Renáta; Ovečka, Miroslav; Pavlovič, Andrej; Slováková, Ľudmila; Blehová, Alžbeta

    2013-01-01

    It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host. PMID:23438585

  6. Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

    Science.gov (United States)

    Singh, Vijayata; Singh, Praveen Kumar; Siddiqui, Adnan; Singh, Subaran; Banday, Zeeshan Zahoor; Nandi, Ashis Kumar

    2016-03-01

    Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

  7. Complete plastid genome sequence of goosegrass (Eleusine indica) and comparison with other Poaceae.

    Science.gov (United States)

    Zhang, Hui; Hall, Nathan; McElroy, J Scott; Lowe, Elijah K; Goertzen, Leslie R

    2017-02-05

    Eleusine indica, also known as goosegrass, is a serious weed in at least 42 countries. In this paper we report the complete plastid genome sequence of goosegrass obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The goosegrass plastome is a circular molecule of 135,151bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 20,919 bases. The large (LSC) and the small (SSC) single-copy regions span 80,667 bases and 12,646 bases, respectively. The plastome of goosegrass has 38.19% GC content and includes 108 unique genes, of which 76 are protein-coding, 28 are transfer RNA, and 4 are ribosomal RNA. The goosegrass plastome sequence was compared to eight other species of Poaceae. Although generally conserved with respect to Poaceae, this genomic resource will be useful for evolutionary studies within this weed species and the genus Eleusine. Copyright © 2016. Published by Elsevier B.V.

  8. The complete plastid genomes of the two 'dinotoms' Durinskia baltica and Kryptoperidinium foliaceum.

    Directory of Open Access Journals (Sweden)

    Behzad Imanian

    2010-05-01

    Full Text Available In one small group of dinoflagellates, photosynthesis is carried out by a tertiary endosymbiont derived from a diatom, giving rise to a complex cell that we collectively refer to as a 'dinotom'. The endosymbiont is separated from its host by a single membrane and retains plastids, mitochondria, a large nucleus, and many other eukaryotic organelles and structures, a level of complexity suggesting an early stage of integration. Although the evolution of these endosymbionts has attracted considerable interest, the plastid genome has not been examined in detail, and indeed no tertiary plastid genome has yet been sequenced.Here we describe the complete plastid genomes of two closely related dinotoms, Durinskia baltica and Kryptoperidinium foliaceum. The D. baltica (116470 bp and K. foliaceum (140426 bp plastid genomes map as circular molecules featuring two large inverted repeats that separate distinct single copy regions. The organization and gene content of the D. baltica plastid closely resemble those of the pennate diatom Phaeodactylum tricornutum. The K. foliaceum plastid genome is much larger, has undergone more reorganization, and encodes a putative tyrosine recombinase (tyrC also found in the plastid genome of the heterokont Heterosigma akashiwo, and two putative serine recombinases (serC1 and serC2 homologous to recombinases encoded by plasmids pCf1 and pCf2 in another pennate diatom, Cylindrotheca fusiformis. The K. foliaceum plastid genome also contains an additional copy of serC1, two degenerate copies of another plasmid-encoded ORF, and two non-coding regions whose sequences closely resemble portions of the pCf1 and pCf2 plasmids.These results suggest that while the plastid genomes of two dinotoms share very similar gene content and genome organization with that of the free-living pennate diatom P. tricornutum, the K. folicaeum plastid genome has absorbed two exogenous plasmids. Whether this took place before or after the tertiary

  9. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2015-01-01

    as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases

  10. Complete plastid genome sequence of Primula sinensis (Primulaceae: structure comparison, sequence variation and evidence for accD transfer to nucleus

    Directory of Open Access Journals (Sweden)

    Tong-Jian Liu

    2016-06-01

    Full Text Available Species-rich genus Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions (25,535 bp were separated by a large single-copy region (82,064 bp and a small single-copy region (17,725 bp. The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis.

  11. Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae: A Comparative Analysis and Phylogenetic Implications.

    Directory of Open Access Journals (Sweden)

    Jie Cai

    Full Text Available Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.

  12. Plastid-bearing sea slugs fix CO2 in the light but do not require photosynthesis to survive

    NARCIS (Netherlands)

    Christa, Gregor; Zimorski, Verena; Woehle, Christian; Tielens, Aloysius G M; Wägele, Heike; Martin, William F; Gould, Sven B

    2014-01-01

    Several sacoglossan sea slugs (Plakobranchoidea) feed upon plastids of large unicellular algae. Four species--called long-term retention (LtR) species--are known to sequester ingested plastids within specialized cells of the digestive gland. There, the stolen plastids (kleptoplasts) remain

  13. Identification and characterization of plastid-type proteins from sequence-attributed features using machine learning

    Science.gov (United States)

    2013-01-01

    Background Plastids are an important component of plant cells, being the site of manufacture and storage of chemical compounds used by the cell, and contain pigments such as those used in photosynthesis, starch synthesis/storage, cell color etc. They are essential organelles of the plant cell, also present in algae. Recent advances in genomic technology and sequencing efforts is generating a huge amount of DNA sequence data every day. The predicted proteome of these genomes needs annotation at a faster pace. In view of this, one such annotation need is to develop an automated system that can distinguish between plastid and non-plastid proteins accurately, and further classify plastid-types based on their functionality. We compared the amino acid compositions of plastid proteins with those of non-plastid ones and found significant differences, which were used as a basis to develop various feature-based prediction models using similarity-search and machine learning. Results In this study, we developed separate Support Vector Machine (SVM) trained classifiers for characterizing the plastids in two steps: first distinguishing the plastid vs. non-plastid proteins, and then classifying the identified plastids into their various types based on their function (chloroplast, chromoplast, etioplast, and amyloplast). Five diverse protein features: amino acid composition, dipeptide composition, the pseudo amino acid composition, Nterminal-Center-Cterminal composition and the protein physicochemical properties are used to develop SVM models. Overall, the dipeptide composition-based module shows the best performance with an accuracy of 86.80% and Matthews Correlation Coefficient (MCC) of 0.74 in phase-I and 78.60% with a MCC of 0.44 in phase-II. On independent test data, this model also performs better with an overall accuracy of 76.58% and 74.97% in phase-I and phase-II, respectively. The similarity-based PSI-BLAST module shows very low performance with about 50% prediction

  14. Interdependency of formation and localisation of the Min complex controls symmetric plastid division.

    Science.gov (United States)

    Maple, Jodi; Møller, Simon G

    2007-10-01

    Plastid division represents a fundamental biological process essential for plant development; however, the molecular basis of symmetric plastid division is unclear. AtMinE1 plays a pivotal role in selection of the plastid division site in concert with AtMinD1. AtMinE1 localises to discrete foci in chloroplasts and interacts with AtMinD1, which shows a similar localisation pattern. Here, we investigate the importance of Min protein complex formation during the chloroplast division process. Dissection of the assembly of the Min protein complex and determination of the interdependency of complex assembly and localisation in planta allow us to present a model of the molecular basis of selection of the division site in plastids. Moreover, functional analysis of AtMinE1 in bacteria demonstrates the level of functional conservation and divergence of the plastidic MinE proteins.

  15. Complete plastid genome of Astragalus mongholicus var. nakaianus (Fabaceae).

    Science.gov (United States)

    Choi, In-Su; Kim, Joo-Hwan; Choi, Byoung-Hee

    2016-07-01

    The first complete plastid genome (plastome) of the largest angiosperm genus, Astragalus, was sequenced for the Korean endangered endemic species A. mongholicus var. nakaianus. Its genome is relatively short (123,633 bp) because it lacks an Inverted Repeat (IR) region. It comprises 110 genes, including four unique rRNAs, 30 tRNAs, and 76 protein-coding genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. The putative pseudogene with abnormal stop codons is atpE. This plastome has no additional inversions when compared with highly variable plastomes from IRLC tribes Fabeae and Trifolieae. Our phylogenetic analysis confirms the non-monophyly of Galegeae.

  16. Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae

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    Monika Szczecińska

    2015-09-01

    Full Text Available Background: The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. Methodology/principal findings: Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161–162 kb, and 21 were duplicated in the inverted repeats (IR region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2 enabled the molecular delimitation of closely-related Pulsatilla

  17. A sea slug’s guide to plastid symbiosis

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    Jan de Vries

    2014-12-01

    Full Text Available Some 140 years ago sea slugs that contained chlorophyll-pigmented granules similar to those of plants were described. While we now understand that these “green granules” are plastids the slugs sequester from siphonaceous algae upon which they feed, surprisingly little is really known about the molecular details that underlie this one of a kind animal-plastid symbiosis. Kleptoplasts are stored in the cytosol of epithelial cells that form the slug’s digestive tubules, and one would guess that the stolen organelles are acquired for their ability to fix carbon, but studies have never really been able to prove that. We also do not know how the organelles are distinguished from the remaining food particles the slugs incorporate with their meal and that include algal mitochondria and nuclei. We know that the ability to store kleptoplasts long-term has evolved only a few times independently among hundreds of sacoglossan species, but we have no idea on what basis. Here we take a closer look at the history of sacoglossan research and discuss recent developments. We argue that, in order to understand what makes this symbiosis work, we will need to focus on the animal’s physiology just as much as we need to commence a detailed analysis of the plastids’ photobiology. Understanding kleptoplasty in sacoglossan slugs requires an unbiased multidisciplinary approach.

  18. Codon adaptation and synonymous substitution rate in diatom plastid genes.

    Science.gov (United States)

    Morton, Brian R; Sorhannus, Ulf; Fox, Martin

    2002-07-01

    Diatom plastid genes are examined with respect to codon adaptation and rates of silent substitution (Ks). It is shown that diatom genes follow the same pattern of codon usage as other plastid genes studied previously. Highly expressed diatom genes display codon adaptation, or a bias toward specific major codons, and these major codons are the same as those in red algae, green algae, and land plants. It is also found that there is a strong correlation between Ks and variation in codon adaptation across diatom genes, providing the first evidence for such a relationship in the algae. It is argued that this finding supports the notion that the correlation arises from selective constraints, not from variation in mutation rate among genes. Finally, the diatom genes are examined with respect to variation in Ks among different synonymous groups. Diatom genes with strong codon adaptation do not show the same variation in synonymous substitution rate among codon groups as the flowering plant psbA gene which, previous studies have shown, has strong codon adaptation but unusually high rates of silent change in certain synonymous groups. The lack of a similar finding in diatoms supports the suggestion that the feature is unique to the flowering plant psbA due to recent relaxations in selective pressure in that lineage.

  19. An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

    Directory of Open Access Journals (Sweden)

    Palmer Jeffrey D

    2006-09-01

    Full Text Available Abstract Background Horizontal gene transfer (HGT to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid. Results Although several genes gave strongly supported conflicting trees under certain conditions, we are confident of HGT in only a single case beyond the rubisco HGT already reported. Most of the conflicts involved near neighbors connected by long branches (e.g. red algae and their secondary hosts, where phylogenetic methods are prone to mislead. However, three genes – clpP, ycf2, and rpl36 – provided strong support for taxa moving far from their organismal position. Further taxon sampling of clpP and ycf2 resulted in rejection of HGT due to long-branch attraction and a serious error in the published plastid genome sequence of Oenothera elata, respectively. A single new case, a bacterial rpl36 gene transferred into the ancestor of the cryptophyte and haptophyte plastids, appears to be a true HGT event. Interestingly, this rpl36 gene is a distantly related paralog of the rpl36 type found in other plastids and most eubacteria. Moreover, the transferred gene has physically replaced the native rpl36 gene, yet flanking genes and intergenic regions show no sign of HGT. This suggests that gene replacement somehow occurred by recombination at the very ends of rpl36, without the level and length of similarity normally expected to support recombination. Conclusion The rpl36 HGT discovered in this study is of considerable interest in terms of both molecular mechanism and phylogeny. The plastid acquisition of a bacterial rpl36 gene via HGT provides the first strong evidence for a sister-group relationship between haptophyte and

  20. An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

    Science.gov (United States)

    Rice, Danny W; Palmer, Jeffrey D

    2006-01-01

    Background Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid. Results Although several genes gave strongly supported conflicting trees under certain conditions, we are confident of HGT in only a single case beyond the rubisco HGT already reported. Most of the conflicts involved near neighbors connected by long branches (e.g. red algae and their secondary hosts), where phylogenetic methods are prone to mislead. However, three genes – clpP, ycf2, and rpl36 – provided strong support for taxa moving far from their organismal position. Further taxon sampling of clpP and ycf2 resulted in rejection of HGT due to long-branch attraction and a serious error in the published plastid genome sequence of Oenothera elata, respectively. A single new case, a bacterial rpl36 gene transferred into the ancestor of the cryptophyte and haptophyte plastids, appears to be a true HGT event. Interestingly, this rpl36 gene is a distantly related paralog of the rpl36 type found in other plastids and most eubacteria. Moreover, the transferred gene has physically replaced the native rpl36 gene, yet flanking genes and intergenic regions show no sign of HGT. This suggests that gene replacement somehow occurred by recombination at the very ends of rpl36, without the level and length of similarity normally expected to support recombination. Conclusion The rpl36 HGT discovered in this study is of considerable interest in terms of both molecular mechanism and phylogeny. The plastid acquisition of a bacterial rpl36 gene via HGT provides the first strong evidence for a sister-group relationship between haptophyte and cryptophyte plastids to the

  1. ATP-dependent molecular chaperones in plastids--More complex than expected.

    Science.gov (United States)

    Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

    2015-09-01

    Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Multiple RNA processing defects and impaired chloroplast function in plants deficient in the organellar protein-only RNase P enzyme.

    Directory of Open Access Journals (Sweden)

    Wenbin Zhou

    Full Text Available Transfer RNA (tRNA precursors undergo endoribonucleolytic processing of their 5' and 3' ends. 5' cleavage of the precursor transcript is performed by ribonuclease P (RNase P. While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs. The plant organellar PRORP (PRORP1 has been characterized to some extent in vitro and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme in vivo by generating stably transformed Arabidopsis plants in which expression of the PRORP1 gene was suppressed by RNA interference (RNAi. PRORP1 knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of PRORP1 knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA and tRNA-Arg(ACG suggests that these two tRNA species limit plastid gene expression in the PRORP1 mutants and, hence, are causally responsible for the mutant phenotype.

  3. Non-photosynthetic plastids as hosts for metabolic engineering

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Behrendorff, James Bruce Yarnton H; Nielsen, Agnieszka Janina Zygadlo

    2018-01-01

    Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive......, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most...... in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis...

  4. Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development

    Science.gov (United States)

    Schaeffer, Scott M.; Christian, Ryan; Castro-Velasquez, Nohely; Hyden, Brennan; Lynch-Holm, Valerie

    2017-01-01

    Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids—chromoplasts—are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant ‘Granny Smith’, carotenoid-predominant ‘Golden Delicious’, and anthocyanin-predominant ‘Top Red Delicious’. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex biochemistry of plastids in perennial non-model systems. PMID:28698906

  5. The plastid and mitochondrial peptidase network and a comprehensive peptidase compendium for Arabidopsis thaliana

    Science.gov (United States)

    Plant plastids and mitochondria have dynamic proteomes. To maintain their protein homeostasis, a proteostasis network containing protein chaperones, peptidases and their substrate recognition factors exists, but many peptidases, their functional connections and substrates are poorly characterized. T...

  6. A contemplation on the secondary origin of green algal and plant plastids

    Directory of Open Access Journals (Sweden)

    Eunsoo Kim

    2014-12-01

    Full Text Available A single origin of plastids and the monophyly of three “primary” plastid-containing groups – the Chloroplastida (or Viridiplantae; green algae+land plants, Rhodophyta, and Glaucophyta – are widely accepted, mainstream hypotheses that form the basis for many comparative evolutionary studies. This “Archaeplastida” hypothesis, however, thus far has not been unambiguously confirmed by phylogenetic studies based on nucleocytoplasmic markers. In view of this as well as other lines of evidence, we suggest the testing of an alternate hypothesis that plastids of the Chloroplastida are of secondary origin. The new hypothesis is in agreement with, or perhaps better explains, existing data, including both the plastidal and nucleocytoplasmic characteristics of the Chloroplastida in comparison to those of other groups.

  7. Plastid Phylogenomic Analyses Resolve Tofieldiaceae as the Root of the Early Diverging Monocot Order Alismatales.

    Science.gov (United States)

    Luo, Yang; Ma, Peng-Fei; Li, Hong-Tao; Yang, Jun-Bo; Wang, Hong; Li, De-Zhu

    2016-04-06

    The predominantly aquatic order Alismatales, which includes approximately 4,500 species within Araceae, Tofieldiaceae, and the core alismatid families, is a key group in investigating the origin and early diversification of monocots. Despite their importance, phylogenetic ambiguity regarding the root of the Alismatales tree precludes answering questions about the early evolution of the order. Here, we sequenced the first complete plastid genomes from three key families in this order:Potamogeton perfoliatus(Potamogetonaceae),Sagittaria lichuanensis(Alismataceae), andTofieldia thibetica(Tofieldiaceae). Each family possesses the typical quadripartite structure, with plastid genome sizes of 156,226, 179,007, and 155,512 bp, respectively. Among them, the plastid genome ofS. lichuanensisis the largest in monocots and the second largest in angiosperms. Like other sequenced Alismatales plastid genomes, all three families generally encode the same 113 genes with similar structure and arrangement. However, we detected 2.4 and 6 kb inversions in the plastid genomes ofSagittariaandPotamogeton, respectively. Further, we assembled a 79 plastid protein-coding gene sequence data matrix of 22 taxa that included the three newly generated plastid genomes plus 19 previously reported ones, which together represent all primary lineages of monocots and outgroups. In plastid phylogenomic analyses using maximum likelihood and Bayesian inference, we show both strong support for Acorales as sister to the remaining monocots and monophyly of Alismatales. More importantly, Tofieldiaceae was resolved as the most basal lineage within Alismatales. These results provide new insights into the evolution of Alismatales as well as the early-diverging monocots as a whole. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  8. The chloroplast min system functions differentially in two specific nongreen plastids in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Peng; Zhang, Jie; Su, Jianbin; Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Wang, Jinfa; Wang, Hongbin

    2013-01-01

    The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS). Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3) and arc12 (VIGS-ALB3) plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3) plants, but organized into multiple rings in parc6 (VIGS-ALB3) and presented fragmented filaments in arc12 (VIGS-ALB3) plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts.

  9. The chloroplast min system functions differentially in two specific nongreen plastids in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Peng Wang

    Full Text Available The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS. Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3 and arc12 (VIGS-ALB3 plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3 plants, but organized into multiple rings in parc6 (VIGS-ALB3 and presented fragmented filaments in arc12 (VIGS-ALB3 plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts.

  10. Analysis of plastid number, size, and distribution in Arabidopsis plants by light and fluorescence microscopy.

    Science.gov (United States)

    Pyke, Kevin

    2011-01-01

    Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis.

  11. Processes for producing polyhydroxybutyrate and related polyhydroxyalkanoates in the plastids of higher plants

    Science.gov (United States)

    Somerville, C.R.; Nawrath, C.; Poirier, Y.

    1997-03-11

    The present invention relates to a process for producing poly-D-(-)-3-hydroxybutyric acid (PHB) and related polyhydroxyalkanoates (PHA) in the plastids of plants. The production of PHB is accomplished by genetically transforming plants with modified genes from microorganisms. The genes encode the enzymes required to synthesize PHB from acetyl-CoA or related metabolites and are fused with additional plant sequences for targeting the enzymes to the plastid. 37 figs.

  12. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  13. Plastid Located WHIRLY1 Enhances the Responsiveness of Arabidopsis Seedlings Toward Abscisic Acid

    Science.gov (United States)

    Isemer, Rena; Krause, Kirsten; Grabe, Nils; Kitahata, Nobutaka; Asami, Tadao; Krupinska, Karin

    2012-01-01

    WHIRLY1 is a protein that can be translocated from the plastids to the nucleus, making it an ideal candidate for communicating information between these two compartments. Mutants of Arabidopsis thaliana lacking WHIRLY1 (why1) were shown to have a reduced sensitivity toward salicylic acid (SA) and abscisic acid (ABA) during germination. Germination assays in the presence of abamine, an inhibitor of ABA biosynthesis, revealed that the effect of SA on germination was in fact caused by a concomitant stimulation of ABA biosynthesis. In order to distinguish whether the plastid or the nuclear isoform of WHIRLY1 is adjusting the responsiveness toward ABA, sequences encoding either the complete WHIRLY1 protein or a truncated form lacking the plastid transit peptide were overexpressed in the why1 mutant background. In plants overexpressing the full-length sequence, WHIRLY1 accumulated in both plastids and the nucleus, whereas in plants overexpressing the truncated sequence, WHIRLY1 accumulated exclusively in the nucleus. Seedlings containing recombinant WHIRLY1 in both compartments were hypersensitive toward ABA. In contrast, seedlings possessing only the nuclear form of WHIRLY1 were as insensitive toward ABA as the why1 mutants. ABA was furthermore shown to lower the rate of germination of wildtype seeds even in the presence of abamine which is known to inhibit the formation of xanthoxin, the plastid located precursor of ABA. From this we conclude that plastid located WHIRLY1 enhances the responsiveness of seeds toward ABA even when ABA is supplied exogenously. PMID:23269926

  14. Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato1

    Science.gov (United States)

    Aoki, Koh; Yamamoto, Miyuki; Wada, Keishiro

    1998-01-01

    Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts. PMID:9765529

  15. Visualisation of plastid outgrowths in potato (Solanum tuberosum L.) tubers by carboxyfluorescein diacetate staining.

    Science.gov (United States)

    Borucki, Wojciech; Bederska, Magdalena; Sujkowska-Rybkowska, Marzena

    2015-05-01

    We describe two types of plastid outgrowths visualised in potato tubers after carboxyfluorescein diacetate staining. Probable esterase activity of the outgrowths has been demonstrated for the first time ever. Plastid outgrowths were observed in the phelloderm and storage parenchyma cells of red potato (S. tuberosum L. cv. Rosalinde) tubers after administration of carboxyfluorescein diacetate stain. Endogenous esterases cleaved off acetic groups to release membrane-unpermeable green fluorescing carboxyfluorescein which accumulated differentially in particular cell compartments. The intensive green fluorescence of carboxyfluorescein exhibited highly branched stromules (stroma-filled plastid tubular projections of the plastid envelope) and allowed distinguishing them within cytoplasmic strands of the phelloderm cells. Stromules (1) were directed towards the nucleus or (2) penetrated the whole cells through the cytoplasmic bands of highly vacuolated phelloderm cells. Those directed towards the nucleus were flattened and adhered to the nuclear envelope. Stromule-like interconnections between two parts of the same plastids (isthmuses) were also observed. We also documented the formation of another type of the stroma-filled plastid outgrowths, referred to here as protrusions, which differed from previously defined stromules in both morphology and esterase activity. Unlike stromules, the protrusions were found to be associated with developmental processes leading to starch accumulation in the storage parenchyma cells. These results strongly suggest that stromules and protrusions exhibit esterase activity. This has been demonstrated for the first time. Morphological and biochemical features as well as possible functions of stromules and protrusions are discussed below.

  16. Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny.

    Science.gov (United States)

    Lee, Sa Mi; Kang, Kyungsu; Chung, Hyungsup; Yoo, Soon Hee; Xu, Xiang Ming; Lee, Seung-Bum; Cheong, Jong-Joo; Daniell, Henry; Kim, Minkyun

    2006-06-30

    The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.

  17. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    Science.gov (United States)

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  18. The plastid-localized pfkB-type carbohydrate kinases FRUCTOKINASE-LIKE 1 and 2 are essential for growth and development of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Gilkerson Jonathan

    2012-07-01

    Full Text Available Abstract Background Transcription of plastid-encoded genes requires two different DNA-dependent RNA polymerases, a nuclear-encoded polymerase (NEP and plastid-encoded polymerase (PEP. Recent studies identified two related pfkB-type carbohydrate kinases, named FRUCTOKINASE-LIKE PROTEIN (FLN1 and FLN2, as components of the thylakoid bound PEP complex in both Arabidopsis thaliana and Sinapis alba (mustard. Additional work demonstrated that RNAi-mediated reduction in FLN expression specifically diminished transcription of PEP-dependent genes. Results Here, we report the characterization of Arabidopsis FLN knockout alleles to examine the contribution of each gene in plant growth, chloroplast development, and in mediating PEP-dependent transcription. We show that fln plants have severe phenotypes with fln1 resulting in an albino phenotype that is seedling lethal without a source of exogenous carbon. In contrast, fln2 plants display chlorosis prior to leaf expansion, but exhibit slow greening, remain autotrophic, can grow to maturity, and set viable seed. fln1 fln2 double mutant analysis reveals haplo-insufficiency, and fln1 fln2 plants have a similar, but more severe phenotype than either single mutant. Normal plastid development in both light and dark requires the FLNs, but surprisingly skotomorphogenesis is unaffected in fln seedlings. Seedlings genetically fln1-1 with dexamethasone-inducible FLN1-HA expression at germination are phenotypically indistinguishable from wild-type. Induction of FLN-HA after 24 hours of germination cannot rescue the mutant phenotype, indicating that the effects of loss of FLN are not always reversible. Examination of chloroplast gene expression in fln1-1 and fln2-1 by qRT-PCR reveals that transcripts of PEP-dependent genes were specifically reduced compared to NEP-dependent genes in both single mutants. Conclusions Our results demonstrate that each FLN protein contributes to wild type growth, and acting additively are

  19. DNA maintenance in plastids and mitochondria of plants

    Directory of Open Access Journals (Sweden)

    Delene J Oldenburg

    2015-10-01

    Full Text Available The DNA molecules in plastids and mitochondria of plants have been studied for over 40 years. Here, we review the data on the circular or linear form, replication, repair, and persistence of the organellar DNA (orgDNA in plants. The bacterial origin of orgDNA appears to have profoundly influenced ideas about the properties of chromosomal DNA molecules in these organelles to the point of dismissing data inconsistent with ideas from the 1970s. When found at all, circular genome-sized molecules comprise a few percent of orgDNA. In cells active in orgDNA replication, most orgDNA is found as linear and branched-linear forms larger than the size of the genome, likely a consequence of a virus-like DNA replication mechanism. In contrast to the stable chromosomal DNA molecules in bacteria and the plant nucleus, the molecular integrity of orgDNA declines during leaf development at a rate that varies among plant species. This decline is attributed to degradation of damaged-but-not-repaired molecules, with a proposed repair cost-saving benefit most evident in grasses. All orgDNA maintenance activities are proposed to occur on the nucleoid tethered to organellar membranes by developmentally-regulated proteins.

  20. Plastid genome structure and loss of photosynthetic ability in the parasitic genus Cuscuta.

    Science.gov (United States)

    Revill, Meredith J W; Stanley, Susan; Hibberd, Julian M

    2005-09-01

    The genus Cuscuta (dodder) is composed of parasitic plants, some species of which appear to be losing the ability to photosynthesize. A molecular phylogeny was constructed using 15 species of Cuscuta in order to assess whether changes in photosynthetic ability and alterations in structure of the plastid genome relate to phylogenetic position within the genus. The molecular phylogeny provides evidence for four major clades within Cuscuta. Although DNA blot analysis showed that Cuscuta species have smaller plastid genomes than tobacco, and that plastome size varied significantly even within one Cuscuta clade, dot blot analysis indicated that the dodders possess homologous sequence to 101 genes from the tobacco plastome. Evidence is provided for significant rates of DNA transfer from plastid to nucleus in Cuscuta. Size and structure of Cuscuta plastid genomes, as well as photosynthetic ability, appear to vary independently of position within the phylogeny, thus supporting the hypothesis that within Cuscuta photosynthetic ability and organization of the plastid genome are changing in an unco-ordinated manner.

  1. Metabolic pathway redundancy within the apicomplexan-dinoflagellate radiation argues against an ancient chromalveolate plastid

    KAUST Repository

    Waller, Ross F.; Gornik, Sebastian G.; Koreny, Ludek; Pain, Arnab

    2015-01-01

    The chromalveolate hypothesis presents an attractively simple explanation for the presence of red algal-derived secondary plastids in 5 major eukaryotic lineages: “chromista” phyla, cryptophytes, haptophytes and ochrophytes; and alveolate phyla, dinoflagellates and apicomplexans. It posits that a single secondary endosymbiotic event occurred in a common ancestor of these diverse groups, and that this ancient plastid has since been maintained by vertical inheritance only. Substantial testing of this hypothesis by molecular phylogenies has, however, consistently failed to provide support for the predicted monophyly of the host organisms that harbour these plastids—the “chromalveolates.” This lack of support does not disprove the chromalveolate hypothesis per se, but rather drives the proposed endosymbiosis deeper into the eukaryotic tree, and requires multiple plastid losses to have occurred within intervening aplastidic lineages. An alternative perspective on plastid evolution is offered by considering the metabolic partnership between the endosymbiont and its host cell. A recent analysis of metabolic pathways in a deep-branching dinoflagellate indicates a high level of pathway redundancy in the common ancestor of apicomplexans and dinoflagellates, and differential losses of these pathways soon after radiation of the major extant lineages. This suggests that vertical inheritance of an ancient plastid in alveolates is highly unlikely as it would necessitate maintenance of redundant pathways over very long evolutionary timescales.

  2. Membrane composition and physiological activity of plastids from an oenothera plastome mutator-induced chloroplast mutant.

    Science.gov (United States)

    Johnson, E M; Sears, B B

    1990-01-01

    Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly.

  3. Metabolic pathway redundancy within the apicomplexan-dinoflagellate radiation argues against an ancient chromalveolate plastid

    KAUST Repository

    Waller, Ross F.

    2015-12-08

    The chromalveolate hypothesis presents an attractively simple explanation for the presence of red algal-derived secondary plastids in 5 major eukaryotic lineages: “chromista” phyla, cryptophytes, haptophytes and ochrophytes; and alveolate phyla, dinoflagellates and apicomplexans. It posits that a single secondary endosymbiotic event occurred in a common ancestor of these diverse groups, and that this ancient plastid has since been maintained by vertical inheritance only. Substantial testing of this hypothesis by molecular phylogenies has, however, consistently failed to provide support for the predicted monophyly of the host organisms that harbour these plastids—the “chromalveolates.” This lack of support does not disprove the chromalveolate hypothesis per se, but rather drives the proposed endosymbiosis deeper into the eukaryotic tree, and requires multiple plastid losses to have occurred within intervening aplastidic lineages. An alternative perspective on plastid evolution is offered by considering the metabolic partnership between the endosymbiont and its host cell. A recent analysis of metabolic pathways in a deep-branching dinoflagellate indicates a high level of pathway redundancy in the common ancestor of apicomplexans and dinoflagellates, and differential losses of these pathways soon after radiation of the major extant lineages. This suggests that vertical inheritance of an ancient plastid in alveolates is highly unlikely as it would necessitate maintenance of redundant pathways over very long evolutionary timescales.

  4. Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

    Science.gov (United States)

    Kanamoto, Hirosuke; Yamashita, Atsushi; Asao, Hiroshi; Okumura, Satoru; Takase, Hisabumi; Hattori, Masahira; Yokota, Akiho; Tomizawa, Ken-Ichi

    2006-04-01

    Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

  5. Comparative ultrastructure of fruit plastids in three genetically diverse genotypes of apple (Malus × domestica Borkh.) during development.

    Science.gov (United States)

    Schaeffer, Scott M; Christian, Ryan; Castro-Velasquez, Nohely; Hyden, Brennan; Lynch-Holm, Valerie; Dhingra, Amit

    2017-10-01

    Comparative ultrastructural developmental time-course analysis has identified discrete stages at which the fruit plastids undergo structural and consequently functional transitions to facilitate subsequent development-guided understanding of the complex plastid biology. Plastids are the defining organelle for a plant cell and are critical for myriad metabolic functions. The role of leaf plastid, chloroplast, is extensively documented; however, fruit plastids-chromoplasts-are poorly understood, especially in the context of the diverse metabolic processes operating in these diverse plant organs. Recently, in a comparative study of the predicted plastid-targeted proteomes across seven plant species, we reported that each plant species is predicted to harbor a unique set of plastid-targeted proteins. However, the temporal and developmental context of these processes remains unknown. In this study, an ultrastructural analysis approach was used to characterize fruit plastids in the epidermal and collenchymal cell layers at 11 developmental timepoints in three genotypes of apple (Malus × domestica Borkh.): chlorophyll-predominant 'Granny Smith', carotenoid-predominant 'Golden Delicious', and anthocyanin-predominant 'Top Red Delicious'. Plastids transitioned from a proplastid-like plastid to a chromoplast-like plastid in epidermis cells, while in the collenchyma cells, they transitioned from a chloroplast-like plastid to a chloro-chromo-amyloplast plastid. Plastids in the collenchyma cells of the three genotypes demonstrated a diverse array of structures and features. This study enabled the identification of discrete developmental stages during which specific functions are most likely being performed by the plastids as indicated by accumulation of plastoglobuli, starch granules, and other sub-organeller structures. Information regarding the metabolically active developmental stages is expected to facilitate biologically relevant omics studies to unravel the complex

  6. The complete nucleotide sequences of the 5 genetically distinct plastid genomes of Oenothera, subsection Oenothera: II. A microevolutionary view using bioinformatics and formal genetic data.

    Science.gov (United States)

    Greiner, Stephan; Wang, Xi; Herrmann, Reinhold G; Rauwolf, Uwe; Mayer, Klaus; Haberer, Georg; Meurer, Jörg

    2008-09-01

    A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.

  7. Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (Apocynaceae)

    Science.gov (United States)

    Shannon C.K. Straub; Richard C. Cronn; Christopher Edwards; Mark Fishbein; Aaron. Liston

    2013-01-01

    Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae...

  8. Plastid ribosomal protein S5 plays a critical role in photosynthesis, plant development, and cold stress tolerance in arabidopsis

    Science.gov (United States)

    Plastid ribosomal proteins (RPs) are essential components for protein synthesis machinery and exert diverse roles in plant growth and development. Mutations in plastid RPs lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood and th...

  9. A complete plastid phylogeny of Daucus – concordance to nuclear results, and markers necessary for phylogenetic resolution

    Science.gov (United States)

    Premise of study: Our purposes were to (1) obtain a well-resolved plastid counterpart to the 94 gene nuclear ortholog gene phylogeny of Arbizu et al. (2014, Amer. J. Bot. 101:1666-1685; and Syst. Bot., in press), and (2) to investigate various classes and numbers of plastid markers necessary for a c...

  10. RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

    Directory of Open Access Journals (Sweden)

    Masaki Odahara

    2015-03-01

    Full Text Available Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

  11. 5S ribosomal RNA database Y2K.

    Science.gov (United States)

    Szymanski, M; Barciszewska, M Z; Barciszewski, J; Erdmann, V A

    2000-01-01

    This paper presents the updated version (Y2K) of the database of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA), http://rose.man/poznan.pl/5SData/index.html. This edition of the database contains 1985primary structures of 5S rRNA and 5S rDNA. They include 60 archaebacterial, 470 eubacterial, 63 plastid, nine mitochondrial and 1383 eukaryotic sequences. The nucleotide sequences of the 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of the source organisms.

  12. The (in)complete organelle genome: exploring the use and nonuse of available technologies for characterizing mitochondrial and plastid chromosomes.

    Science.gov (United States)

    Sanitá Lima, Matheus; Woods, Laura C; Cartwright, Matthew W; Smith, David Roy

    2016-11-01

    Not long ago, scientists paid dearly in time, money and skill for every nucleotide that they sequenced. Today, DNA sequencing technologies epitomize the slogan 'faster, easier, cheaper and more', and in many ways, sequencing an entire genome has become routine, even for the smallest laboratory groups. This is especially true for mitochondrial and plastid genomes. Given their relatively small sizes and high copy numbers per cell, organelle DNAs are currently among the most highly sequenced kind of chromosome. But accurately characterizing an organelle genome and the information it encodes can require much more than DNA sequencing and bioinformatics analyses. Organelle genomes can be surprisingly complex and can exhibit convoluted and unconventional modes of gene expression. Unravelling this complexity can demand a wide assortment of experiments, from pulsed-field gel electrophoresis to Southern and Northern blots to RNA analyses. Here, we show that it is exactly these types of 'complementary' analyses that are often lacking from contemporary organelle genome papers, particularly short 'genome announcement' articles. Consequently, crucial and interesting features of organelle chromosomes are going undescribed, which could ultimately lead to a poor understanding and even a misrepresentation of these genomes and the genes they express. High-throughput sequencing and bioinformatics have made it easy to sequence and assemble entire chromosomes, but they should not be used as a substitute for or at the expense of other types of genomic characterization methods. © 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

  13. A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids

    Czech Academy of Sciences Publication Activity Database

    Janouškovec, J.; Horák, A.; Oborník, Miroslav; Lukeš, Julius; Keeling, P. J.

    2010-01-01

    Roč. 107, č. 24 (2010), s. 10949-10954 ISSN 0027-8424 R&D Projects: GA AV ČR IAA601410907 Institutional research plan: CEZ:AV0Z60220518 Keywords : Apicomplexa * Chromera velia * CCMP3155 * plastid evolution * chloroplast genome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.771, year: 2010

  14. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

    Science.gov (United States)

    Gutensohn, Michael; Orlova, Irina; Nguyen, Thuong T H; Davidovich-Rikanati, Rachel; Ferruzzi, Mario G; Sitrit, Yaron; Lewinsohn, Efraim; Pichersky, Eran; Dudareva, Natalia

    2013-08-01

    Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  15. YCF45 protein, usually associated with plastids, is targeted into the mitochondrion of Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Týč, Jiří; Long, Shaojun; Jirků, Milan; Lukeš, Julius

    2010-01-01

    Roč. 173, č. 1 (2010), s. 43-47 ISSN 0166-6851 R&D Projects: GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trypanosoma * Plastid * Mitochondrion * Targeting * YCF45 * Horizontal gene transfer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.875, year: 2010

  16. Ultrastructural study on dynamics of lipid bodies and plastids during ripening of chili pepper fruits.

    Science.gov (United States)

    Liu, Lin

    2013-03-01

    Dynamics of lipid bodies and plastids in chili pepper fruits during ripening were investigated by means of transmission electron microscopy. Mesocarp of chili pepper fruits consists of collenchyma, normal parenchyma, and huge celled parenchyma. In mature green fruits, plastids contain numerous thylakoids that are well organized into grana in collenchyma, a strikingly huge amount of starch and irregularly organized thylakoids in normal parenchyma, and simple tubes rather than thylakoids in huge celled parenchyma. These morphological features suggest that plastids are chloroplasts in collenchyma, chloroamyloplasts in normal parenchyma, proplastids in huge celled parenchyma. As fruits ripen to red, plastids in all cell types convert to chromoplasts and, concomitantly, lipid bodies accumulate in both cytoplasm and chromoplasts. Cytosolic lipid bodies are lined up in a regular layer adjacent to plasma membrane. The cytosolic lipid body consists of a core surrounded by a membrane. The core is comprised of a more electron-dense central part enclosed by a slightly less electron-dense peripheral layer. Plastidial lipid bodies in collenchyma, normal parenchyma, and endodermis initiate as plastoglobuli, which in turn convert to rod-like structures. Therefore, plastidial lipid bodies are more dynamic than cytosolic lipid bodies. Both cytosolic and plastidial lipid bodies contain rich unsaturated lipids. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  18. Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

    Directory of Open Access Journals (Sweden)

    Preetinder K Dhanoa

    Full Text Available BACKGROUND: Tail-anchored (TA proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34 and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9. Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie

  19. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    KAUST Repository

    Huerlimann, Roger

    2015-07-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  20. Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants.

    Science.gov (United States)

    Rogalski, Marcelo; Carrer, Helaine

    2011-06-01

    The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes. However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  1. Are algal genes in nonphotosynthetic protists evidence of historical plastid endosymbioses?

    Directory of Open Access Journals (Sweden)

    Tian Jing

    2009-10-01

    Full Text Available Abstract Background How photosynthetic organelles, or plastids, were acquired by diverse eukaryotes is among the most hotly debated topics in broad scale eukaryotic evolution. The history of plastid endosymbioses commonly is interpreted under the "chromalveolate" hypothesis, which requires numerous plastid losses from certain heterotrophic groups that now are entirely aplastidic. In this context, discoveries of putatively algal genes in plastid-lacking protists have been cited as evidence of gene transfer from a photosynthetic endosymbiont that subsequently was lost completely. Here we examine this evidence, as it pertains to the chromalveolate hypothesis, through genome-level statistical analyses of similarity scores from queries with two diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, and two aplastidic sister taxa, Phytophthora ramorum and P. sojae. Results Contingency tests of specific predictions of the chromalveolate model find no evidence for an unusual red algal contribution to Phytophthora genomes, nor that putative cyanobacterial sequences that are present entered these genomes through a red algal endosymbiosis. Examination of genes unrelated to plastid function provide extraordinarily significant support for both of these predictions in diatoms, the control group where a red endosymbiosis is known to have occurred, but none of that support is present in genes specifically conserved between diatoms and oomycetes. In addition, we uncovered a strong association between overall sequence similarities among taxa and relative sizes of genomic data sets in numbers of genes. Conclusion Signal from "algal" genes in oomycete genomes is inconsistent with the chromalveolate hypothesis, and better explained by alternative models of sequence and genome evolution. Combined with the numerous sources of intragenomic phylogenetic conflict characterized previously, our results underscore the potential to be mislead by a posteriori

  2. The complete plastid genome of Bunge's pine Pinus bungeana (Pinaceae).

    Science.gov (United States)

    Li, Zhong-Hu; Zhu, Juan; Yang, Yi-Xin; Yang, Jie; He, Jing-Wen; Zhao, Gui-Fang

    2016-07-01

    The complete nucleotide sequence of Bunge's pine Pinus bungeana Zucc. ex Endl. chloroplast genome (cp DNA) was determined in this study. The cpDNA was 117 861 bp in length, containing a pair of 475 bp inverted repeat regions (IRa and IRb), which were separated by large and small single copy regions (LSC and SSC) of 65 373 and 51 538 bp, respectively. The cpDNA contained 111 genes, including 71 protein-coding genes (71 PCG species), 4 ribosomal RNA genes (4 rRNA species) and 36 tRNA genes (32 tRNA species). In these genes, 13 harbored a single intron and 1 (ycf3) contained a couple of introns. The overall AT content of Bunge's pine cpDNA is 61.2%, while the corresponding values of the LSC, SSC and IR regions are 61.9%, 60.2% and 62.5%, respectively. A phylogenetic reconstruction based on the maximum parsimony analysis suggested that all the sampled Pinus species clustered a monophyletic clade and have a high bootstrap support, and the cpDNA of P. bungeana is closely related to that of congeneric P. gerardiana.

  3. The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

    Science.gov (United States)

    Fujiwara, Makoto T; Yasuzawa, Mana; Kojo, Kei H; Niwa, Yasuo; Abe, Tomoko; Yoshida, Shigeo; Nakano, Takeshi; Itoh, Ryuuichi D

    2018-01-01

    Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division

  4. Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

    Science.gov (United States)

    Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

    2008-08-22

    CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

  5. A phylogenetic analysis of the genus Psathyrostachys (Poaceae) based on one nuclear gene, three plastid genes, and morphology

    DEFF Research Database (Denmark)

    Petersen, Gitte; Seberg, Ole; Baden, Claus

    2004-01-01

    A phylogenetic analysis of the small, Central Asian genus Psathyrostachys Nevski is presented. The analysis is based on morphological characters and nucleotide sequence data from one nuclear gene, DMC1, and three plastid genes, rbcL, rpoA, and rpoC2. Separate analyses of the three data partitions...... (morphology, nuclear sequences, and plastid sequences) result in mostly congruent trees. The plastid and nuclear sequences produce completely congruent trees, and only the trees based on plastid sequences and morphological characters are incongruent. Combined analysis of all data results in a fairly well......-resolved strict consensus tree: Ps. rupestris is the sister to the remaining species, which are divided into two clades: one including Ps. fragilis and Ps. caduca, the other including Ps. juncea, Ps. huashanica, Ps. lanuginosa, Ps. stoloniformis, and Ps. kronenburgii. Pubescent culms and more than 20 mm long...

  6. Plastid Transformation in the Monocotyledonous Cereal Crop, Rice (Oryza sativa) and Transmission of Transgenes to Their Progeny

    OpenAIRE

    Lee, Sa Mi; Kang, Kyungsu; Chung, Hyunsup; Yoo, Soon Hee; Xu, Xiang Ming; Lee, Seung-Bum; Cheong, Jong-Joo; Daniell, Henry; Kim, Minkyun

    2006-01-01

    The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stab...

  7. Contrasting Patterns of Nucleotide Substitution Rates Provide Insight into Dynamic Evolution of Plastid and Mitochondrial Genomes of Geranium

    OpenAIRE

    Park, Seongjun; Ruhlman, Tracey A.; Weng, Mao-Lun; Hajrah, Nahid H.; Sabir, Jamal S.M.; Jansen, Robert K.

    2017-01-01

    Abstract Geraniaceae have emerged as a model system for investigating the causes and consequences of variation in plastid and mitochondrial genomes. Incredible structural variation in plastid genomes (plastomes) and highly accelerated evolutionary rates have been reported in selected lineages and functional groups of genes in both plastomes and mitochondrial genomes (mitogenomes), and these phenomena have been implicated in cytonuclear incompatibility. Previous organelle genome studies have i...

  8. Hedysarum L. (Fabaceae: Hedysareae) Is Not Monophyletic - Evidence from Phylogenetic Analyses Based on Five Nuclear and Five Plastid Sequences.

    Science.gov (United States)

    Liu, Pei-Liang; Wen, Jun; Duan, Lei; Arslan, Emine; Ertuğrul, Kuddisi; Chang, Zhao-Yang

    2017-01-01

    The legume family (Fabaceae) exhibits a high level of species diversity and evolutionary success worldwide. Previous phylogenetic studies of the genus Hedysarum L. (Fabaceae: Hedysareae) showed that the nuclear and the plastid topologies might be incongruent, and the systematic position of the Hedysarum sect. Stracheya clade was uncertain. In this study, phylogenetic relationships of Hedysarum were investigated based on the nuclear ITS, ETS, PGDH, SQD1, TRPT and the plastid psbA-trnH, trnC-petN, trnL-trnF, trnS-trnG, petN-psbM sequences. Both nuclear and plastid data support two major lineages in Hedysarum: the Hedysarum s.s. clade and the Sartoria clade. In the nuclear tree, Hedysarum is biphyletic with the Hedysarum s.s. clade sister to the Corethrodendron + Eversmannia + Greuteria + Onobrychis clade (the CEGO clade), whereas the Sartoria clade is sister to the genus Taverniera DC. In the plastid tree, Hedysarum is monophyletic and sister to Taverniera. The incongruent position of the Hedysarum s.s. clade between the nuclear and plastid trees may be best explained by a chloroplast capture hypothesis via introgression. The Hedysarum sect. Stracheya clade is resolved as sister to the H. sect. Hedysarum clade in both nuclear and plastid trees, and our analyses support merging Stracheya into Hedysarum. Based on our new evidence from multiple sequences, Hedysarum is not monophyletic, and its generic delimitation needs to be reconsidered.

  9. Hedysarum L. (Fabaceae: Hedysareae) Is Not Monophyletic – Evidence from Phylogenetic Analyses Based on Five Nuclear and Five Plastid Sequences

    Science.gov (United States)

    Liu, Pei-Liang; Wen, Jun; Duan, Lei; Arslan, Emine; Ertuğrul, Kuddisi; Chang, Zhao-Yang

    2017-01-01

    The legume family (Fabaceae) exhibits a high level of species diversity and evolutionary success worldwide. Previous phylogenetic studies of the genus Hedysarum L. (Fabaceae: Hedysareae) showed that the nuclear and the plastid topologies might be incongruent, and the systematic position of the Hedysarum sect. Stracheya clade was uncertain. In this study, phylogenetic relationships of Hedysarum were investigated based on the nuclear ITS, ETS, PGDH, SQD1, TRPT and the plastid psbA-trnH, trnC-petN, trnL-trnF, trnS-trnG, petN-psbM sequences. Both nuclear and plastid data support two major lineages in Hedysarum: the Hedysarum s.s. clade and the Sartoria clade. In the nuclear tree, Hedysarum is biphyletic with the Hedysarum s.s. clade sister to the Corethrodendron + Eversmannia + Greuteria + Onobrychis clade (the CEGO clade), whereas the Sartoria clade is sister to the genus Taverniera DC. In the plastid tree, Hedysarum is monophyletic and sister to Taverniera. The incongruent position of the Hedysarum s.s. clade between the nuclear and plastid trees may be best explained by a chloroplast capture hypothesis via introgression. The Hedysarum sect. Stracheya clade is resolved as sister to the H. sect. Hedysarum clade in both nuclear and plastid trees, and our analyses support merging Stracheya into Hedysarum. Based on our new evidence from multiple sequences, Hedysarum is not monophyletic, and its generic delimitation needs to be reconsidered. PMID:28122062

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

    Science.gov (United States)

    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.

  11. Plastid transformation in lettuce (Lactuca sativa L.) by biolistic DNA delivery.

    Science.gov (United States)

    Ruhlman, Tracey A

    2014-01-01

    The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa L.). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide an ideal platform for the production of biopharmaceuticals.

  12. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  13. Cuscuta europaea plastid apparatus in various developmental stages: localization of THF1 protein.

    Science.gov (United States)

    Švubová, Renáta; Ovečka, Miroslav; Pavlovič, Andrej; Slováková, L'udmila; Blehová, Alžbeta

    2013-05-01

    It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in "get together" tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.

  14. Plastid Genome Evolution in the Early-Diverging Legume Subfamily Cercidoideae (Fabaceae

    Directory of Open Access Journals (Sweden)

    Yin-Huan Wang

    2018-02-01

    Full Text Available The subfamily Cercidoideae is an early-branching legume lineage, which consists of 13 genera distributed in the tropical and warm temperate Northern Hemisphere. A previous study detected two plastid genomic variations in this subfamily, but the limited taxon sampling left the overall plastid genome (plastome diversification across the subfamily unaddressed, and phylogenetic relationships within this clade remained unresolved. Here, we assembled eight plastomes from seven Cercidoideae genera and conducted phylogenomic-comparative analyses in a broad evolutionary framework across legumes. The plastomes of Cercidoideae all exhibited a typical quadripartite structure with a conserved gene content typical of most angiosperm plastomes. Plastome size ranged from 151,705 to 165,416 bp, mainly due to the expansion and contraction of inverted repeat (IR regions. The order of genes varied due to the occurrence of several inversions. In Tylosema species, a plastome with a 29-bp IR-mediated inversion was found to coexist with a canonical-type plastome, and the abundance of the two arrangements of isomeric molecules differed between individuals. Complete plastome data were much more efficient at resolving intergeneric relationships of Cercidoideae than the previously used selection of only a few plastid or nuclear loci. In sum, our study revealed novel insights into the structural diversification of plastomes in an early-branching legume lineage, and, thus, into the evolutionary trajectories of legume plastomes in general.

  15. A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

    Science.gov (United States)

    Johnson, E M; Schnabelrauch, L S; Sears, B B

    1991-01-01

    Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

  16. Expansion of inverted repeat does not decrease substitution rates in Pelargonium plastid genomes.

    Science.gov (United States)

    Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

    2017-04-01

    For species with minor inverted repeat (IR) boundary changes in the plastid genome (plastome), nucleotide substitution rates were previously shown to be lower in the IR than the single copy regions (SC). However, the impact of large-scale IR expansion/contraction on plastid nucleotide substitution rates among closely related species remains unclear. We included plastomes from 22 Pelargonium species, including eight newly sequenced genomes, and used both pairwise and model-based comparisons to investigate the impact of the IR on sequence evolution in plastids. Ten types of plastome organization with different inversions or IR boundary changes were identified in Pelargonium. Inclusion in the IR was not sufficient to explain the variation of nucleotide substitution rates. Instead, the rate heterogeneity in Pelargonium plastomes was a mixture of locus-specific, lineage-specific and IR-dependent effects. Our study of Pelargonium plastomes that vary in IR length and gene content demonstrates that the evolutionary consequences of retaining these repeats are more complicated than previously suggested. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  17. A plastid gene phylogeny of the non-photosynthetic parasitic Orobanche (Orobanchaceae) and related genera.

    Science.gov (United States)

    Park, Jeong-Mi; Manen, Jean-François; Colwell, Alison E; Schneeweiss, Gerald M

    2008-07-01

    The phylogenetic relationships of the non-photosynthetic Orobanche sensu lato (Orobanchaceae), which includes some of the economically most important parasitic weeds, remain insufficiently understood and controversial. This concerns both the phylogenetic relationships within the genus, in particular its monophyly or lack thereof, and the relationships to other holoparasitic genera such as Cistanche or Conopholis. Here we present the first comprehensive phylogenetic study of this group based on a region from the plastid genome (rps2 gene). Although substitution rates appear to be elevated compared to the photosynthetic members of Orobanchaceae, relationships among the major lineages Cistanche, Conopholis plus Epifagus, Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch., B. himalaica Hook. f. & Thomson, B. hookeri Walp. plus B. strobilacea A. Gray, and Orobanche s. l. remain unresolved. Resolution within Orobanche, however, is much better. In agreement with morphological, cytological and other molecular phylogenetic evidence, five lineages, corresponding to the four traditionally recognised sections (Gymnocaulis, Myzorrhiza, Orobanche, Trionychon) and O. latisquama Reut. ex Boiss. (of sect. Orobanche), can be distinguished. A combined analysis of plastid rps2 and nuclear ITS sequences of the holoparasitic genera results in more resolved and better supported trees, although the relationships among Orobanche s. l., Cistanche, and the clade including the remaining genera is unresolved. Therefore, rps2 is a marker from the plastid genome that is well-suited to be used in combination with other already established nuclear markers for resolving generic relationships of Orobanche and related genera.

  18. A plastid gene phylogeny of the non-photosynthetic parasitic Orobanche (Orobanchaceae) and related genera

    Science.gov (United States)

    Park, J.-M.; Manen, J.-F.; Colwell, A.E.; Schneeweiss, G.M.

    2008-01-01

    The phylogenetic relationships of the non-photosynthetic Orobanche sensu lato (Orobanchaceae), which includes some of the economically most important parasitic weeds, remain insufficiently understood and controversial. This concerns both the phylogenetic relationships within the genus, in particular its monophyly or lack thereof, and the relationships to other holoparasitic genera such as Cistanche or Conopholis. Here we present the first comprehensive phylogenetic study of this group based on a region from the plastid genome (rps2 gene). Although substitution rates appear to be elevated compared to the photosynthetic members of Orobanchaceae, relationships among the major lineages Cistanche, Conopholis plus Epifagus, Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch., B. himalaica Hook. f. & Thomson, B. hookeri Walp. plus B. strobilacea A. Gray, and Orobanche s. l. remain unresolved. Resolution within Orobanche, however, is much better. In agreement with morphological, cytological and other molecular phylogenetic evidence, five lineages, corresponding to the four traditionally recognised sections (Gymnocaulis, Myzorrhiza, Orobanche, Trionychon) and O. latisquama Reut. ex Boiss. (of sect. Orobanche), can be distinguished. A combined analysis of plastid rps2 and nuclear ITS sequences of the holoparasitic genera results in more resolved and better supported trees, although the relationships among Orobanche s. l., Cistanche, and the clade including the remaining genera is unresolved. Therefore, rps2 is a marker from the plastid genome that is well-suited to be used in combination with other already established nuclear markers for resolving generic relationships of Orobanche and related genera. ?? 2008 The Botanical Society of Japan and Springer.

  19. Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

    Science.gov (United States)

    Mujer, C V; Andrews, D L; Manhart, J R; Pierce, S K; Rumpho, M E

    1996-10-29

    The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic

  20. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  1. Reconstruction of molecular phylogeny of closely related Amorphophallus species of India using plastid DNA marker and fingerprinting approaches.

    Science.gov (United States)

    Gholave, Avinash R; Pawar, Kiran D; Yadav, Shrirang R; Bapat, Vishwas A; Jadhav, Jyoti P

    2017-01-01

    Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL , matK , trnH - psbA , trnLC - trnLD , their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus , Conophallus and Amorphophallus . In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK , trnH - psbA , trnLC - trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL .

  2. Horizontal Transfer of DNA from the Mitochondrial to the Plastid Genome and Its Subsequent Evolution in Milkweeds (Apocynaceae)

    Science.gov (United States)

    Straub, Shannon C.K.; Cronn, Richard C.; Edwards, Christopher; Fishbein, Mark; Liston, Aaron

    2013-01-01

    Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2–rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes. PMID:24029811

  3. Membrane Composition and Physiological Activity of Plastids from an Oenothera Plastome Mutator-Induced Chloroplast Mutant 1

    Science.gov (United States)

    Johnson, Ellen M.; Sears, Barbara B.

    1990-01-01

    Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly. PMID:16667256

  4. Split photosystem protein, linear-mapping topology, and growth of structural complexity in the plastid genome of chromera velia

    KAUST Repository

    Janouškovec, Jan

    2013-08-22

    The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here, we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulsed-field gel electrophoresis and polymerase chain reaction, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions, and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function. © The Author 2013.

  5. Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

    Directory of Open Access Journals (Sweden)

    Shin-Yi Shyu

    2013-12-01

    Full Text Available Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998 performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989 were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.

  6. Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

    Science.gov (United States)

    Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

    2018-05-14

    Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

  7. Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre.

    Science.gov (United States)

    Hartmann, Manuela; Grob, Carolina; Scanlan, David J; Martin, Adrian P; Burkill, Peter H; Zubkov, Mikhail V

    2011-11-01

    The smallest phototrophic protists (protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4-29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (Pprotists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition - predation on phosphorus-rich bacterioplankton cells. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  8. Complete nucleotide sequence of the Oenothera elata plastid chromosome, representing plastome I of the five distinguishable euoenothera plastomes.

    Science.gov (United States)

    Hupfer, H; Swiatek, M; Hornung, S; Herrmann, R G; Maier, R M; Chiu, W L; Sears, B

    2000-05-01

    We describe the 159,443-bp [corrected] sequence of the plastid chromosome of Oenothera elata (evening primrose). The Oe. elata plastid chromosome represents type I of the five genetically distinguishable basic plastomes found in the subsection Euoenothera. The genus Oenothera provides an ideal system in which to address fundamental questions regarding the functional integration of the compartmentalised genetic system characteristic of the eukaryotic cell. Its highly developed taxonomy and genetics, together with a favourable combination of features in its genetic structure (interspecific fertility, stable heterozygous progeny, biparental transmission of organelles, and the phenomenon of complex heterozygosity), allow facile exchanges of nuclei, plastids and mitochondria, as well as individual chromosome pairs, between species. The resulting hybrids or cybrids are usually viable and fertile, but can display various forms of developmental disturbance.

  9. Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species, little divergence.

    Science.gov (United States)

    Turner, Barbara; Paun, Ovidiu; Munzinger, Jérôme; Chase, Mark W; Samuel, Rosabelle

    2016-06-01

    Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species. Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices. The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species. In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with plastid DNA did not help to resolve the phylogenetic tree

  10. Systematics and plastid genome evolution of the cryptically photosynthetic parasitic plant genus Cuscuta (Convolvulaceae

    Directory of Open Access Journals (Sweden)

    Kuehl Jennifer V

    2007-12-01

    Full Text Available Abstract Background The genus Cuscuta L. (Convolvulaceae, commonly known as dodders, are epiphytic vines that invade the stems of their host with haustorial feeding structures at the points of contact. Although they lack expanded leaves, some species are noticeably chlorophyllous, especially as seedlings and in maturing fruits. Some species are reported as crop pests of worldwide distribution, whereas others are extremely rare and have local distributions and apparent niche specificity. A strong phylogenetic framework for this large genus is essential to understand the interesting ecological, morphological and molecular phenomena that occur within these parasites in an evolutionary context. Results Here we present a well-supported phylogeny of Cuscuta using sequences of the nuclear ribosomal internal transcribed spacer and plastid rps2, rbcL and matK from representatives across most of the taxonomic diversity of the genus. We use the phylogeny to interpret morphological and plastid genome evolution within the genus. At least three currently recognized taxonomic sections are not monophyletic and subgenus Cuscuta is unequivocally paraphyletic. Plastid genes are extremely variable with regards to evolutionary constraint, with rbcL exhibiting even higher levels of purifying selection in Cuscuta than photosynthetic relatives. Nuclear genome size is highly variable within Cuscuta, particularly within subgenus Grammica, and in some cases may indicate the existence of cryptic species in this large clade of morphologically similar species. Conclusion Some morphological characters traditionally used to define major taxonomic splits within Cuscuta are homoplastic and are of limited use in defining true evolutionary groups. Chloroplast genome evolution seems to have evolved in a punctuated fashion, with episodes of loss involving suites of genes or tRNAs followed by stabilization of gene content in major clades. Nearly all species of Cuscuta retain some

  11. Systematics and plastid genome evolution of the cryptically photosynthetic parasitic plant genus Cuscuta (Convolvulaceae).

    Science.gov (United States)

    McNeal, Joel R; Arumugunathan, Kathiravetpilla; Kuehl, Jennifer V; Boore, Jeffrey L; Depamphilis, Claude W

    2007-12-13

    The genus Cuscuta L. (Convolvulaceae), commonly known as dodders, are epiphytic vines that invade the stems of their host with haustorial feeding structures at the points of contact. Although they lack expanded leaves, some species are noticeably chlorophyllous, especially as seedlings and in maturing fruits. Some species are reported as crop pests of worldwide distribution, whereas others are extremely rare and have local distributions and apparent niche specificity. A strong phylogenetic framework for this large genus is essential to understand the interesting ecological, morphological and molecular phenomena that occur within these parasites in an evolutionary context. Here we present a well-supported phylogeny of Cuscuta using sequences of the nuclear ribosomal internal transcribed spacer and plastid rps2, rbcL and matK from representatives across most of the taxonomic diversity of the genus. We use the phylogeny to interpret morphological and plastid genome evolution within the genus. At least three currently recognized taxonomic sections are not monophyletic and subgenus Cuscuta is unequivocally paraphyletic. Plastid genes are extremely variable with regards to evolutionary constraint, with rbcL exhibiting even higher levels of purifying selection in Cuscuta than photosynthetic relatives. Nuclear genome size is highly variable within Cuscuta, particularly within subgenus Grammica, and in some cases may indicate the existence of cryptic species in this large clade of morphologically similar species. Some morphological characters traditionally used to define major taxonomic splits within Cuscuta are homoplastic and are of limited use in defining true evolutionary groups. Chloroplast genome evolution seems to have evolved in a punctuated fashion, with episodes of loss involving suites of genes or tRNAs followed by stabilization of gene content in major clades. Nearly all species of Cuscuta retain some photosynthetic ability, most likely for nutrient

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Rate variation in parasitic plants: correlated and uncorrelated patterns among plastid genes of different function

    Directory of Open Access Journals (Sweden)

    dePamphilis Claude W

    2005-02-01

    Full Text Available Abstract Background The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon. Results Phylogenetic reconstructions of relative rates of sequence evolution for three plastid genes (rbcL, matK and rps2 show significant rate heterogeneity among lineages and among genes. Many of the non-photosynthetic plants have increases in both synonymous and nonsynonymous rates, indicating that both (1 selection is relaxed, and (2 there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not always immediate upon loss of photosynthesis. Overall there is a poor correlation of synonymous and nonsynonymous rates. There is, however, a strong correlation of synonymous rates across the 3 genes studied and the lineage-speccific pattern for each gene is strikingly similar. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is a weaker correlation across genes for nonsynonymous rates. Here the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene. Conclusions The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many (though not all non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors.

  14. Genome BLAST distance phylogenies inferred from whole plastid and whole mitochondrion genome sequences

    Directory of Open Access Journals (Sweden)

    Holland Barbara R

    2006-07-01

    Full Text Available Abstract Background Phylogenetic methods which do not rely on multiple sequence alignments are important tools in inferring trees directly from completely sequenced genomes. Here, we extend the recently described Genome BLAST Distance Phylogeny (GBDP strategy to compute phylogenetic trees from all completely sequenced plastid genomes currently available and from a selection of mitochondrial genomes representing the major eukaryotic lineages. BLASTN, TBLASTX, or combinations of both are used to locate high-scoring segment pairs (HSPs between two sequences from which pairwise similarities and distances are computed in different ways resulting in a total of 96 GBDP variants. The suitability of these distance formulae for phylogeny reconstruction is directly estimated by computing a recently described measure of "treelikeness", the so-called δ value, from the respective distance matrices. Additionally, we compare the trees inferred from these matrices using UPGMA, NJ, BIONJ, FastME, or STC, respectively, with the NCBI taxonomy tree of the taxa under study. Results Our results indicate that, at this taxonomic level, plastid genomes are much more valuable for inferring phylogenies than are mitochondrial genomes, and that distances based on breakpoints are of little use. Distances based on the proportion of "matched" HSP length to average genome length were best for tree estimation. Additionally we found that using TBLASTX instead of BLASTN and, particularly, combining TBLASTX and BLASTN leads to a small but significant increase in accuracy. Other factors do not significantly affect the phylogenetic outcome. The BIONJ algorithm results in phylogenies most in accordance with the current NCBI taxonomy, with NJ and FastME performing insignificantly worse, and STC performing as well if applied to high quality distance matrices. δ values are found to be a reliable predictor of phylogenetic accuracy. Conclusion Using the most treelike distance matrices, as

  15. The mitochondrial and plastid genomes of Volvox carteri: bloated molecules rich in repetitive DNA

    Directory of Open Access Journals (Sweden)

    Lee Robert W

    2009-03-01

    Full Text Available Abstract Background The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA and plastid DNA (ptDNA sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence. Results We sequenced 30 and 420 kilobases (kb of the mitochondrial and plastid genomes of V. carteri, respectively – resulting in partial assemblies of these genomes. The mitochondrial genome is the most bloated green-algal mtDNA observed to date: ~61% of the sequence is noncoding, most of which is comprised of short palindromic repeats spread throughout the intergenic and intronic regions. The plastid genome is the largest (>420 kb and most expanded (>80% noncoding ptDNA sequence yet discovered, with a myriad of palindromic repeats in the noncoding regions, which have a similar size and secondary structure to those of the mtDNA. We found that 15 kb (~0.01% of the nuclear genome are homologous to the palindromic elements of the mtDNA, and 50 kb (~0.05% are homologous to those of the ptDNA. Conclusion Selfish elements in the form of short palindromic repeats have propagated in the V. carteri mtDNA and ptDNA, resulting in the distension of these genomes. Copies of these same repeats are also found in a small fraction of the nucDNA, but appear to be inert in this

  16. Plastid genome evolution across the genus Cuscuta (Convolvulaceae): two clades within subgenus Grammica exhibit extensive gene loss.

    Science.gov (United States)

    Braukmann, Thomas; Kuzmina, Maria; Stefanovic, Sasa

    2013-02-01

    The genus Cuscuta (Convolvulaceae, the morning glory family) is one of the most intensely studied lineages of parasitic plants. Whole plastome sequencing of four Cuscuta species has demonstrated changes to both plastid gene content and structure. The presence of photosynthetic genes under purifying selection indicates that Cuscuta is cryptically photosynthetic. However, the tempo and mode of plastid genome evolution across the diversity of this group (~200 species) remain largely unknown. A comparative investigation of plastid genome content, grounded within a phylogenetic framework, was conducted using a slot-blot Southern hybridization approach. Cuscuta was extensively sampled (~56% of species), including groups previously suggested to possess more altered plastomes compared with other members of this genus. A total of 56 probes derived from all categories of protein-coding genes, typically found within the plastomes of flowering plants, were used. The results indicate that two clades within subgenus Grammica (clades 'O' and 'K') exhibit substantially more plastid gene loss relative to other members of Cuscuta. All surveyed members of the 'O' clade show extensive losses of plastid genes from every category of genes typically found in the plastome, including otherwise highly conserved small and large ribosomal subunits. The extent of plastid gene losses within this clade is similar in magnitude to that observed previously in some non-asterid holoparasites, in which the very presence of a plastome has been questioned. The 'K' clade also exhibits considerable loss of plastid genes. Unlike in the 'O' clade, in which all species seem to be affected, the losses in clade 'K' progress phylogenetically, following a pattern consistent with the Evolutionary Transition Series hypothesis. This clade presents an ideal opportunity to study the reduction of the plastome of parasites 'in action'. The widespread plastid gene loss in these two clades is hypothesized to be a

  17. Nuclear and plastid DNAs from the binucleate dinoflagellates Glenodinium (Peridinium) foliaceum and Peridinium balticum.

    Science.gov (United States)

    Kite, G C; Rothschild, L J; Dodge, J D

    1988-01-01

    The binucleate dinoflagellates Glenodinium (Peridinium) foliaceum Stein and Peridinium balticum (Levander) Lemmermann were found to contain two major buoyant density classes of DNA. The heavier peak (1.730 g/cm3) was derived from the "dinokaryotic" nucleus and the lighter peak (1.706 g/cm3) from the "endosymbiont" nucleus and this allowed for the fractionation of G. foliaceum DNA in CsCl/EtBr density gradients. An initial CsCl/Hoechst Dye gradient removed a minor A-T rich satellite species which was identified as plastid DNA with a size of about 100-106 kb. Analysis of the nuclear DNA by agarose gel electrophoresis and renaturation studies showed that the endosymbiont nucleus lacked amplified gene-sized DNA molecules, however, this nucleus did have a comparatively high level of DNA. The total amount of DNA per cell and the relative contributions of the two nuclei appeared to vary between two strains of G. foliaceum (75 pg/cell in CCAP strain and 58 pg in UTEX strain). The only strain of P. balticum examined contained 73 pg cell. These results are discussed in relation to the status and possible functioning of the endosymbiont nucleus and the idea that these dinoflagellates provide model systems with which to study the evolution of plastids.

  18. Chaperone-like properties of tobacco plastid thioredoxins f and m

    Science.gov (United States)

    Sanz-Barrio, Ruth; Fernández-San Millán, Alicia; Carballeda, Jon; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Farran, Inmaculada

    2012-01-01

    Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein. PMID:21948853

  19. Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers.

    Science.gov (United States)

    Kelchner, Scot A

    2013-05-01

    Bamboos are large perennial grasses of temperate and tropical forests worldwide. Two general growth forms exist: the economically and ecologically important woody bamboos (tribes Arundinarieae and Bambuseae), and the understory herbaceous bamboos (tribe Olyreae). Evolutionary relationships among the 1400+described species have been difficult to resolve with confidence. Comparative analysis of bamboo plastid (chloroplast) DNA has revealed three to five major lineages that show distinct biogeographic distributions. Taxon sampling across tribes and subtribes has been incomplete and most published data sets include a relatively small number of nucleotide characters. Branching order among lineages is often poorly supported, and in more than one study herbaceous bamboos form a clade within the woody bamboos. In this paper, the Bamboo Phylogeny Group presents the most complete phylogeny estimation to date of bamboo tribes and subtribes using 6.7 kb of coding and noncoding sequence data and 37 microstructural characters from the chloroplast genome. Quality of data is assessed, as is the possibility of long branch attraction, the degree of character conflict at key nodes in the tree, and the legitimacy of three alternative hypotheses of relationship. Four major plastid lineages are recognized: temperate woody, paleotropical woody, neotropical woody, and herbaceous bamboos. Woody bamboos are resolved as paraphyletic with respect to Olyreae but SH tests cannot reject monophyly of woody species (Arundinarieae+Bambuseae). Published by Elsevier Inc.

  20. Diversification of Rosaceae since the Late Cretaceous based on plastid phylogenomics.

    Science.gov (United States)

    Zhang, Shu-Dong; Jin, Jian-Jun; Chen, Si-Yun; Chase, Mark W; Soltis, Douglas E; Li, Hong-Tao; Yang, Jun-Bo; Li, De-Zhu; Yi, Ting-Shuang

    2017-05-01

    Phylogenetic relationships in Rosaceae have long been problematic because of frequent hybridisation, apomixis and presumed rapid radiation, and their historical diversification has not been clarified. With 87 genera representing all subfamilies and tribes of Rosaceae and six of the other eight families of Rosales (outgroups), we analysed 130 newly sequenced plastomes together with 12 from GenBank in an attempt to reconstruct deep relationships and reveal temporal diversification of this family. Our results highlight the importance of improving sequence alignment and the use of appropriate substitution models in plastid phylogenomics. Three subfamilies and 16 tribes (as previously delimited) were strongly supported as monophyletic, and their relationships were fully resolved and strongly supported at most nodes. Rosaceae were estimated to have originated during the Late Cretaceous with evidence for rapid diversification events during several geological periods. The major lineages rapidly diversified in warm and wet habits during the Late Cretaceous, and the rapid diversification of genera from the early Oligocene onwards occurred in colder and drier environments. Plastid phylogenomics offers new and important insights into deep phylogenetic relationships and the diversification history of Rosaceae. The robust phylogenetic backbone and time estimates we provide establish a framework for future comparative studies on rosaceous evolution. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  1. Citrus plastid-related gene profiling based on expressed sequence tag analyses

    Directory of Open Access Journals (Sweden)

    Tercilio Calsa Jr.

    2007-01-01

    Full Text Available Plastid-related sequences, derived from putative nuclear or plastome genes, were searched in a large collection of expressed sequence tags (ESTs and genomic sequences from the Citrus Biotechnology initiative in Brazil. The identified putative Citrus chloroplast gene sequences were compared to those from Arabidopsis, Eucalyptus and Pinus. Differential expression profiling for plastid-directed nuclear-encoded proteins and photosynthesis-related gene expression variation between Citrus sinensis and Citrus reticulata, when inoculated or not with Xylella fastidiosa, were also analyzed. Presumed Citrus plastome regions were more similar to Eucalyptus. Some putative genes appeared to be preferentially expressed in vegetative tissues (leaves and bark or in reproductive organs (flowers and fruits. Genes preferentially expressed in fruit and flower may be associated with hypothetical physiological functions. Expression pattern clustering analysis suggested that photosynthesis- and carbon fixation-related genes appeared to be up- or down-regulated in a resistant or susceptible Citrus species after Xylella inoculation in comparison to non-infected controls, generating novel information which may be helpful to develop novel genetic manipulation strategies to control Citrus variegated chlorosis (CVC.

  2. Structural changes in plastids of developing Splachnum ampullaceum sporophytes and relationship to odour production.

    Science.gov (United States)

    McCuaig, B; Dufour, S C; Raguso, R A; Bhatt, A P; Marino, P

    2015-03-01

    Many mosses of the family Splachnaceae are entomophilous and rely on flies for spore dispersal. Splachnum ampullaceum produces a yellow- or pink-coloured hypophysis that releases volatile compounds, attracting flies to the mature moss. The biosynthetic sources of the visual and aromatic cues within the hypophysis have not been identified, and may be either symbiotic cyanobacteria or chromoplasts that break down lipids into volatile compounds. Here, we used transmission electron microscopy and gas chromatography-mass spectrometry (GC-MS) to investigate the sources of these attractants, focusing on different tissues and stages of maturation. Microscopy revealed an abundance of plastids within the hypophysis, while no symbiotic bacteria were observed. During plant maturation, plastids differentiated from amyloplasts with large starch granules to photosynthetic chloroplasts and finally to chromoplasts with lipid accumulations. We used GC-MS to identify over 50 volatile organic compounds from mature sporophytes including short-chain oxygenated compounds, unsaturated irregular terpenoids, fatty acid-derived 6- and 8-carbon alcohols and ketones, and the aromatic compounds acetophenone and p-cresol. The hypophysis showed localised production of pungent volatiles, mainly short-chain fermentation compounds and p-cresol. Some of these volatiles have been shown to be produced from lipid oxidase degradation of linolenic acid within chromoplasts. However, other compounds (such as cyclohexanecarboxylic acid esters) may have a microbial origin. Further investigation is necessary to identify the origin of fly attractants in these mosses. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  3. Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

    Energy Technology Data Exchange (ETDEWEB)

    Heemskerk, J.W.M.; Heinz, E. (Univ. of Hamburg (West Germany)); Storz, T.; Schmidt, R.R. (Univ. of Konstanz (West Germany))

    1990-08-01

    Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-(6-{sup 3}H)galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5{prime}-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG.

  4. A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

    Science.gov (United States)

    Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

    2000-01-01

    The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

  5. Structural changes in plastids of developing Splachnum ampullaceum sporophytes and relationship to odour production

    Science.gov (United States)

    McCuaig, B.; Dufour, S. C.; Raguso, R. A.; Bhatt, A. P.; Marino, P.

    2014-01-01

    Many mosses of the family Splachnaceae are entomophilous and rely on flies for spore dispersal. Splachnum ampullaceum produces a yellow- or pink-coloured hypophysis that releases volatile compounds, attracting flies to the mature moss. The biosynthetic sources of the visual and aromatic cues within the hypophysis have not been identified, and may be either symbiotic cyanobacteria or chromoplasts that break down lipids into volatile compounds. Here, we used transmission electron microscopy and gas chromatography-mass spectrometry (GC-MS) to investigate the sources of these attractants, focusing on different tissues and stages of maturation. Microscopy revealed an abundance of plastids within the hypophysis, while no symbiotic bacteria were observed. During plant maturation, plastids differentiated from amyloplasts with large starch granules to photosynthetic chloroplasts and finally to chromoplasts with lipid accumulations. We used GC-MS to identify over 50 volatile organic compounds from mature sporophytes including short-chain oxygenated compounds, unsaturated irregular terpenoids, fatty acid-derived 6- and 8-carbon alcohols and ketones, and the aromatic compounds acetophenone and p-cresol. The hypophysis showed localised production of pungent volatiles, mainly short-chain fermentation compounds and p-cresol. Some of these volatiles have been shown to be produced from lipid oxidase degradation of linolenic acid within chromoplasts. However, other compounds (such as cyclohexanecarboxylic acid esters) may have a microbial origin. Further investigation is necessary to identify the origin of fly attractants in these mosses. PMID:25213550

  6. Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites.

    Science.gov (United States)

    Andrabi, Syed Bilal Ahmad; Tahara, Michiru; Matsubara, Ryuma; Toyama, Tomoko; Aonuma, Hiroka; Sakakibara, Hitoshi; Suematsu, Makoto; Tanabe, Kazuyuki; Nozaki, Tomoyoshi; Nagamune, Kisaburo

    2018-02-01

    Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

    International Nuclear Information System (INIS)

    Heemskerk, J.W.M.; Heinz, E.; Storz, T.; Schmidt, R.R.

    1990-01-01

    Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6- 3 H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5'-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG

  8. Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences.

    Science.gov (United States)

    Lyra, Goia de M; Costa, Emmanuelle da S; de Jesus, Priscila B; de Matos, João Carlos G; Caires, Taiara A; Oliveira, Mariana C; Oliveira, Eurico C; Xi, Zhenxiang; Nunes, José Marcos de C; Davis, Charles C

    2015-04-01

    Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well-supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G. domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae. © 2015 Phycological Society of America.

  9. Investigation into the Physiological Significance of the Phytohormone Abscisic Acid in Perkinsus marinus, an Oyster Parasite Harboring a Nonphotosynthetic Plastid.

    Science.gov (United States)

    Sakamoto, Hirokazu; Suzuki, Shigeo; Nagamune, Kisaburo; Kita, Kiyoshi; Matsuzaki, Motomichi

    2017-07-01

    Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid-harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus. © 2016 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

  10. Shine-dalgarno sequences play an essential role in the translation of plastid mRNAs in tobacco

    DEFF Research Database (Denmark)

    Scharff, Lars; Ehrnthaler, Miriam; Janowski, Marcin

    2017-01-01

    SD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations...

  11. A robust and well-resolved phylogeny of Bactridinae (Arecaceae) based on plastid and nuclear DNA sequences

    DEFF Research Database (Denmark)

    Eiserhardt, Wolf L.; Pintaud, Jean-Christophe; Asmussen-Lange, Conny

    as well as most of the currently accepted infrageneric taxa and recently proposed informal groups. Analyses are based on five plastid DNA regions (matK, trnQ-rps16, rps16 intron, trnD-trnT, trnL-trnF) and three nuclear markers (PRK, RPB2, ITS). A combined dataset was analysed with likelihood and parsimony...

  12. Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays.

    NARCIS (Netherlands)

    Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

    2007-01-01

    Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

  13. Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays

    NARCIS (Netherlands)

    Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

    2007-01-01

    Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

  14. Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

    Science.gov (United States)

    Park, Jeong-Mi; Manen, Jean-François; Schneeweiss, Gerald M

    2007-06-01

    Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.

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

    Czech Academy of Sciences Publication Activity Database

    Burki, F.; Flegontov, Pavel; Oborník, Miroslav; Cihlář, Jaromír; Pain, A.; Lukeš, Julius; Keeling, P. J.

    2012-01-01

    Roč. 4, č. 6 (2012), s. 738-747 ISSN 1759-6653 R&D Projects: GA ČR GAP506/12/1522; GA ČR GBP501/12/G055 Institutional support: RVO:60077344 Keywords : Endosymbiotic gene transfer * plastid evolution * protist * algae * chromera Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.759, year: 2012

  16. RNA transcription in isolated chloroplasts during senescence and rejuvenation of intact cotyledons of CUCURBITA PEPO L. (ZUCCHINI)

    International Nuclear Information System (INIS)

    Mishev, K.; Ananiev, E.; Denev, L.; Radeva, G.

    2006-01-01

    RNA transcription was studied in intact chloroplasts isolated from cotyledons of Cucurbita pepoL. (zucchini) during their growth and development including natural senescence and rejuvenation. Rejuvenation of cotyledons was studied after decapitation of the epicotyl above the senescing yellow cotyledons. Maximal incorporation of [32P] UTP into overall chloroplast RNA was measured two days after exposure of seedlings to light (day 6 th after the onset of germination), followed by a gradual decrease reaching minimal values at the age of 25-28 days when cotyledons began to yellow and eventually die. Rejuvenation of cotyledons completely restored chloroplast RNA synthesis and fifteen days after decapitation (at the age of 40 days), the values of chloroplast transcription even exceeded that of the maximal transcriptional activity in young cotyledons. Inhibitory analysis with tagetitoxin (a specific inhibitor of plastid encoded chloroplast RNA polymerase (PEP)) showed that in young and rejuvenated cotyledons about 85% of chloroplast RNA polymerase activity was due to PEP and only 15% corresponded to the nuclear encoded plastid RNA polymerase (NEP). Definite regions of two chloroplast encoded genes were amplified by means of PCR technique using specific DNA primers for Rubisco large subunit gene (rbcL) and the housekeeping gene for chloroplast 16S rRNA as well as chloroplast DNA as a template. The appropriate lengths of the amplified DNA fragments were checked by restriction analysis

  17. Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling

    International Nuclear Information System (INIS)

    Park, Joon-Heum; Jung, Sunyo

    2017-01-01

    In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. - Highlights: • Two modes of photooxidation by carotenoid and tetrapyrrole biosynthetic inhibitors. • We examine differential alterations in chloroplast function and plastid signaling. • NF and OF cause differential alterations in chloroplast ultrastructure and function. • Photooxidation coordinates photosynthetic gene expression from nucleus and plastid.

  18. Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

    Directory of Open Access Journals (Sweden)

    Seabra Ana R

    2010-08-01

    Full Text Available Abstract Background Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS, occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. Results This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2 in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. Conclusions This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate

  19. Tree ferns: monophyletic groups and their relationships as revealed by four protein-coding plastid loci.

    Science.gov (United States)

    Korall, Petra; Pryer, Kathleen M; Metzgar, Jordan S; Schneider, Harald; Conant, David S

    2006-06-01

    Tree ferns are a well-established clade within leptosporangiate ferns. Most of the 600 species (in seven families and 13 genera) are arborescent, but considerable morphological variability exists, spanning the giant scaly tree ferns (Cyatheaceae), the low, erect plants (Plagiogyriaceae), and the diminutive endemics of the Guayana Highlands (Hymenophyllopsidaceae). In this study, we investigate phylogenetic relationships within tree ferns based on analyses of four protein-coding, plastid loci (atpA, atpB, rbcL, and rps4). Our results reveal four well-supported clades, with genera of Dicksoniaceae (sensu ) interspersed among them: (A) (Loxomataceae, (Culcita, Plagiogyriaceae)), (B) (Calochlaena, (Dicksonia, Lophosoriaceae)), (C) Cibotium, and (D) Cyatheaceae, with Hymenophyllopsidaceae nested within. How these four groups are related to one other, to Thyrsopteris, or to Metaxyaceae is weakly supported. Our results show that Dicksoniaceae and Cyatheaceae, as currently recognised, are not monophyletic and new circumscriptions for these families are needed.

  20. Authentication of Botanical Origin in Herbal Teas by Plastid Noncoding DNA Length Polymorphisms.

    Science.gov (United States)

    Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

    2015-07-01

    The aim of this study was to develop a DNA barcode assay to authenticate the botanical origin of herbal teas. To reach this aim, we tested the efficiency of a PCR-capillary electrophoresis (PCR-CE) approach on commercial herbal tea samples using two noncoding plastid barcodes, the trnL intron and the intergenic spacer between trnL and trnF. Barcode DNA length polymorphisms proved successful in authenticating the species origin of herbal teas. We verified the validity of our approach by sequencing species-specific barcode amplicons from herbal tea samples. Moreover, we displayed the utility of PCR-CE assays coupled with sequencing to identify the origin of undeclared plant material in herbal tea samples. The PCR-CE assays proposed in this work can be applied as routine tests for the verification of botanical origin in herbal teas and can be extended to authenticate all types of herbal foodstuffs.

  1. Plastids features and transfer cells occurrence in the phloem of Portulaca mucronata and P. hirsutissima (Portulacaceae

    Directory of Open Access Journals (Sweden)

    Maria E. Maranhão Estelita

    2014-01-01

    Full Text Available The species of the Portulacaceae of the Serra do Cipó, State of Minas Gerais, Brasil, were studied. In Portulaca mucronata and P. hirsutissima transfer cells are companion and phloem parenchyma cells; they have the same secondary cell wall features, that is, short papillate protuberances which are uniformly distributed around the primary cell wall. These features are similar in both species but they are very distinct from others referred in the literature; this could be useful in Taxonomy. The phloem plastids have a globular protein crystalloid, surrounded by proteinaceous filaments. In P. hirsutissima few starch grains may also be present, and this occurrence is considered primitive in the phylogenetic scale. These features agree with presumptive evolution of those of leaf structure: P. hirsutissima has C3 photosynthesis structure, and P. mucronata C4 or Kranz anatomy.

  2. Expression of chloroperoxidase from Pseudomonas pyrrocinia in tobacco plastids for fungal resistance.

    Science.gov (United States)

    Ruhlman, Tracey A; Rajasekaran, Kanniah; Cary, Jeffrey W

    2014-11-01

    The chloroperoxidase (cpo) gene from Pseudomonas pyrrocinia was transformed into the plastid genome (plastome) of Nicotiana tabacum var. Petit Havana and transplastomic lines were compared with a nuclear transformant for the same gene. Southern analysis confirmed integration in the plastome and western blotting confirmed the presence of the chloroperoxidase protein (CPO) in higher abundance in transplastomic plants than in cpo nuclear transformants. Northern analysis of primary plastome transformants for cpo showed 15-fold higher transcript abundance than in the nuclear transformant, yet this extent of enhancement was not observed in western blot, enzyme or bioassay, indicating a bottleneck at the post-transcriptional level. Representative plants from the two transplastomic lines showed resistance to fungal pathogens in vitro (Aspergillus flavus, Fusarium verticillioides, and Verticillium dahliae) and in planta (Alternaria alternata). Published by Elsevier Ireland Ltd.

  3. Minimally destructive sampling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes.

    Science.gov (United States)

    Hughey, Jeffery R; Gabrielson, Paul W; Rohmer, Laurence; Tortolani, Jacquie; Silva, Mayra; Miller, Kathy Ann; Young, Joel D; Martell, Craig; Ruediger, Erik

    2014-06-04

    Plant species, including algae and fungi, are based on type specimens to which the name of a taxon is permanently attached. Applying a scientific name to any specimen therefore requires demonstrating correspondence between the type and that specimen. Traditionally, identifications are based on morpho-anatomical characters, but recently systematists are using DNA sequence data. These studies are flawed if the DNA is isolated from misidentified modern specimens. We propose a genome-based solution. Using 4 × 4 mm(2) of material from type specimens, we assembled 14 plastid and 15 mitochondrial genomes attributed to the red algae Pyropia perforata, Py. fucicola, and Py. kanakaensis. The chloroplast genomes were fairly conserved, but the mitochondrial genomes differed significantly among populations in content and length. Complete genomes are attainable from 19(th) and early 20(th) century type specimens; this validates the effort and cost of their curation as well as supports the practice of the type method.

  4. Minimally destructive sampling of type specimens of Pyropia (Bangiales, Rhodophyta) recovers complete plastid and mitochondrial genomes

    Science.gov (United States)

    Hughey, Jeffery R.; Gabrielson, Paul W.; Rohmer, Laurence; Tortolani, Jacquie; Silva, Mayra; Miller, Kathy Ann; Young, Joel D.; Martell, Craig; Ruediger, Erik

    2014-01-01

    Plant species, including algae and fungi, are based on type specimens to which the name of a taxon is permanently attached. Applying a scientific name to any specimen therefore requires demonstrating correspondence between the type and that specimen. Traditionally, identifications are based on morpho-anatomical characters, but recently systematists are using DNA sequence data. These studies are flawed if the DNA is isolated from misidentified modern specimens. We propose a genome-based solution. Using 4 × 4 mm2 of material from type specimens, we assembled 14 plastid and 15 mitochondrial genomes attributed to the red algae Pyropia perforata, Py. fucicola, and Py. kanakaensis. The chloroplast genomes were fairly conserved, but the mitochondrial genomes differed significantly among populations in content and length. Complete genomes are attainable from 19th and early 20th century type specimens; this validates the effort and cost of their curation as well as supports the practice of the type method. PMID:24894641

  5. Molecular phylogeny of Gavilea (Chloraeinae: Orchidaceae) using plastid and nuclear markers.

    Science.gov (United States)

    Chemisquy, M Amelia; Morrone, Osvaldo

    2012-03-01

    A phylogenetic analysis is provided for 70% of the representatives of genus Gavilea, as well as for several species of the remaining genera of subtribe Chloraeinae: Bipinnula, Chloraea and Geoblasta. Sequences from the plastid markers rpoC1, matK-trnK and atpB-rbcL and the nuclear marker ITS, were analyzed using Maximum Parsimony and Bayesian Inference. Monophyly of subtribe Chloraeinae was confirmed, as well as its position inside tribe Cranichideae. Neither Chloraea nor Bipinnula were recovered as monophyletic. Gavilea turned out polyphyletic, with Chloraeachica embedded in the genus while Gavilea supralabellata was related to Chloraea and might be a hybrid between both genera. None of the two sections of Gavilea were monophyletic, and the topologies obtained do not suggest a new division of the genus. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Science.gov (United States)

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  7. Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences.

    Science.gov (United States)

    Manen, Jean-François; Habashi, Christine; Jeanmonod, Daniel; Park, Jeong-Mi; Schneeweiss, Gerald M

    2004-11-01

    The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.

  8. Production of high levels of poly-3-hydroxybutyrate in plastids of Camelina sativa seeds.

    Science.gov (United States)

    Malik, Meghna R; Yang, Wenyu; Patterson, Nii; Tang, Jihong; Wellinghoff, Rachel L; Preuss, Mary L; Burkitt, Claire; Sharma, Nirmala; Ji, Yuanyuan; Jez, Joseph M; Peoples, Oliver P; Jaworski, Jan G; Cahoon, Edgar B; Snell, Kristi D

    2015-06-01

    Poly-3-hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed-specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N-terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T1 seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T2 seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight-averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T4 seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed-based platform for PHB production. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Directory of Open Access Journals (Sweden)

    Linda J Savage

    Full Text Available The Chloroplast 2010 Project (http://www.plastid.msu.edu/ identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/. Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles

  10. The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution, Molecular Marker Identification, and Phylogenetic Implications in Asterids

    Science.gov (United States)

    Ku, Chuan; Chung, Wan-Chia; Chen, Ling-Ling; Kuo, Chih-Horng

    2013-01-01

    The Madagascar periwinkle ( Catharanthus roseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C . roseus , which could be applied to other C . roseus -related studies. The C . roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffea arabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepias syriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C . roseus -specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C . roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported. PMID:23825699

  11. Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue.

    Science.gov (United States)

    Egea, Isabel; Bian, Wanping; Barsan, Cristina; Jauneau, Alain; Pech, Jean-Claude; Latché, Alain; Li, Zhengguo; Chervin, Christian

    2011-08-01

    There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase. Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices. At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts. These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.

  12. Paternal inheritance of plastid-encoded transgenes in Petunia hybrida in the greenhouse and under field conditions.

    Science.gov (United States)

    Horn, Patricia; Nausch, Henrik; Baars, Susanne; Schmidtke, Jörg; Schmidt, Kerstin; Schneider, Anja; Leister, Dario; Broer, Inge

    2017-12-01

    As already demonstrated in greenhouse trials, outcrossing of transgenic plants can be drastically reduced via transgene integration into the plastid. We verified this result in the field with Petunia , for which the highest paternal leakage has been observed. The variety white 115 (W115) served as recipient and Pink Wave (PW) and the transplastomic variant PW T16, encoding the uid A reporter gene, as pollen donor. While manual pollination in the greenhouse led to over 90% hybrids for both crossings, the transgenic donor resulted only in 2% hybrids in the field. Nevertheless paternal leakage was detected in one case which proves that paternal inheritance of plastid-located transgenes is possible under artificial conditions. In the greenhouse, paternal leakage occurred in a frequency comparable to published results. As expected natural pollination reduced the hybrid formation in the field from 90 to 7.6% and the transgenic donor did not result in any hybrid.

  13. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  14. Contrasting Patterns of Nucleotide Substitution Rates Provide Insight into Dynamic Evolution of Plastid and Mitochondrial Genomes of Geranium.

    Science.gov (United States)

    Park, Seongjun; Ruhlman, Tracey A; Weng, Mao-Lun; Hajrah, Nahid H; Sabir, Jamal S M; Jansen, Robert K

    2017-06-01

    Geraniaceae have emerged as a model system for investigating the causes and consequences of variation in plastid and mitochondrial genomes. Incredible structural variation in plastid genomes (plastomes) and highly accelerated evolutionary rates have been reported in selected lineages and functional groups of genes in both plastomes and mitochondrial genomes (mitogenomes), and these phenomena have been implicated in cytonuclear incompatibility. Previous organelle genome studies have included limited sampling of Geranium, the largest genus in the family with over 400 species. This study reports on rates and patterns of nucleotide substitutions in plastomes and mitogenomes of 17 species of Geranium and representatives of other Geraniaceae. As detected across other angiosperms, substitution rates in the plastome are 3.5 times higher than the mitogenome in most Geranium. However, in the branch leading to Geranium brycei/Geranium incanum mitochondrial genes experienced significantly higher dN and dS than plastid genes, a pattern that has only been detected in one other angiosperm. Furthermore, rate accelerations differ in the two organelle genomes with plastomes having increased dN and mitogenomes with increased dS. In the Geranium phaeum/Geranium reflexum clade, duplicate copies of clpP and rpoA genes that experienced asymmetric rate divergence were detected in the single copy region of the plastome. In the case of rpoA, the branch leading to G. phaeum/G. reflexum experienced positive selection or relaxation of purifying selection. Finally, the evolution of acetyl-CoA carboxylase is unusual in Geraniaceae because it is only the second angiosperm family where both prokaryotic and eukaryotic ACCases functionally coexist in the plastid. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. The effect of chromate on the synthesis of plastid pigments and lipoquinones in Zea mays L. seedlings

    Directory of Open Access Journals (Sweden)

    Zbigniew Krupa

    2014-01-01

    Full Text Available Maize plants cultivated on nutrient solutions containing increasing amounts of CrO42- (from 10-7 to 10-4 M showed growth inhibition, strong reduction of the root system and an increase of dry matter of leaves. The highest accumulation of chromium in plant organs appeared in the roots. Chromate taken up into leaves caused changes in the content of plastid pigments and lipoquinones similar, in general, to those in senescent plants.

  16. 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).

  17. Evolutionary and biotechnology implications of plastid genome variation in the inverted-repeat-lacking clade of legumes.

    Science.gov (United States)

    Sabir, Jamal; Schwarz, Erika; Ellison, Nicholas; Zhang, Jin; Baeshen, Nabih A; Mutwakil, Muhammed; Jansen, Robert; Ruhlman, Tracey

    2014-08-01

    Land plant plastid genomes (plastomes) provide a tractable model for evolutionary study in that they are relatively compact and gene dense. Among the groups that display an appropriate level of variation for structural features, the inverted-repeat-lacking clade (IRLC) of papilionoid legumes presents the potential to advance general understanding of the mechanisms of genomic evolution. Here, are presented six complete plastome sequences from economically important species of the IRLC, a lineage previously represented by only five completed plastomes. A number of characters are compared across the IRLC including gene retention and divergence, synteny, repeat structure and functional gene transfer to the nucleus. The loss of clpP intron 2 was identified in one newly sequenced member of IRLC, Glycyrrhiza glabra. Using deeply sequenced nuclear transcriptomes from two species helped clarify the nature of the functional transfer of accD to the nucleus in Trifolium, which likely occurred in the lineage leading to subgenus Trifolium. Legumes are second only to cereal crops in agricultural importance based on area harvested and total production. Genetic improvement via plastid transformation of IRLC crop species is an appealing proposition. Comparative analyses of intergenic spacer regions emphasize the need for complete genome sequences for developing transformation vectors for plastid genetic engineering of legume crops. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  18. Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

    Science.gov (United States)

    Bellot, Sidonie; Cusimano, Natalie; Luo, Shixiao; Sun, Guiling; Zarre, Shahin; Gröger, Andreas; Temsch, Eva; Renner, Susanne S

    2016-08-03

    Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

    Science.gov (United States)

    Park, Joon-Heum; Jung, Sunyo

    2017-01-22

    In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Paternal inheritance of plastid-encoded transgenes in Petunia hybrida in the greenhouse and under field conditions

    Directory of Open Access Journals (Sweden)

    Patricia Horn

    2017-12-01

    Full Text Available As already demonstrated in greenhouse trials, outcrossing of transgenic plants can be drastically reduced via transgene integration into the plastid. We verified this result in the field with Petunia, for which the highest paternal leakage has been observed. The variety white 115 (W115 served as recipient and Pink Wave (PW and the transplastomic variant PW T16, encoding the uidA reporter gene, as pollen donor. While manual pollination in the greenhouse led to over 90% hybrids for both crossings, the transgenic donor resulted only in 2% hybrids in the field. Nevertheless paternal leakage was detected in one case which proves that paternal inheritance of plastid-located transgenes is possible under artificial conditions. In the greenhouse, paternal leakage occurred in a frequency comparable to published results. As expected natural pollination reduced the hybrid formation in the field from 90 to 7.6% and the transgenic donor did not result in any hybrid. Keywords: Paternal plastid inheritance, Transgene confinement, Greenhouse, Field trial, Pollen mediated gene flow

  1. Phylogeny and systematics of the brake fern genus Pteris (Pteridaceae) based on molecular (plastid and nuclear) and morphological evidence.

    Science.gov (United States)

    Zhang, Liang; Zhang, Li-Bing

    2018-01-01

    The brake fern genus Pteris belongs to Pteridaceae subfamily Pteridoideae. It is one of the largest fern genera and has been estimated to contain 200-250 species distributed on all continents except Antarctica. Previous studies were either based on plastid data only or based on both plastid and nuclear data but the sampling was small. In addition, an infrageneric classification of Pteris based on morphological and molecular evidence has not been available yet. In the present study, based on molecular data of eight plastid markers and one nuclear marker (gapCp) of 256 accessions representing ca. 178 species of Pteris, we reconstruct a global phylogeny of Pteris. The 15 major clades identified earlier are recovered here and we further identified a new major clade. Our nuclear phylogeny recovered 11 of these 16 major clades, seven of which are strongly supported. The inclusion of Schizostege in Pteris is confirmed for the first time. Based on the newly reconstructed phylogeny and evidence from morphology, distribution and/or ecology, we classify Pteris into three subgenera: P. subg. Pteris, P. subg. Campteria, and P. subg. Platyzoma. The former two are further divided into three and 12 sections, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers.

    Science.gov (United States)

    Gao, Lei; Zhou, Yuan; Wang, Zhi-Wei; Su, Ying-Juan; Wang, Ting

    2011-04-13

    The rpoB-psbZ (BZ) region of some fern plastid genomes (plastomes) has been noted to go through considerable genomic changes. Unraveling its evolutionary dynamics across all fern lineages will lead to clarify the fundamental process shaping fern plastome structure and organization. A total of 24 fern BZ sequences were investigated with taxon sampling covering all the extant fern orders. We found that: (i) a tree fern Plagiogyria japonica contained a novel gene order that can be generated from either the ancestral Angiopteris type or the derived Adiantum type via a single inversion; (ii) the trnY-trnE intergenic spacer (IGS) of the filmy fern Vandenboschia radicans was expanded 3-fold due to the tandem 27-bp repeats which showed strong sequence similarity with the anticodon domain of trnY; (iii) the trnY-trnE IGSs of two horsetail ferns Equisetum ramosissimum and E. arvense underwent an unprecedented 5-kb long expansion, more than a quarter of which was consisted of a single type of direct repeats also relevant to the trnY anticodon domain; and (iv) ycf66 has independently lost at least four times in ferns. Our results provided fresh insights into the evolutionary process of fern BZ regions. The intermediate BZ gene order was not detected, supporting that the Adiantum type was generated by two inversions occurring in pairs. The occurrence of Vandenboschia 27-bp repeats represents the first evidence of partial tRNA gene duplication in fern plastomes. Repeats potentially forming a stem-loop structure play major roles in the expansion of the trnY-trnE IGS.

  3. Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers

    Directory of Open Access Journals (Sweden)

    Su Ying-Juan

    2011-04-01

    Full Text Available Abstract Background The rpoB-psbZ (BZ region of some fern plastid genomes (plastomes has been noted to go through considerable genomic changes. Unraveling its evolutionary dynamics across all fern lineages will lead to clarify the fundamental process shaping fern plastome structure and organization. Results A total of 24 fern BZ sequences were investigated with taxon sampling covering all the extant fern orders. We found that: (i a tree fern Plagiogyria japonica contained a novel gene order that can be generated from either the ancestral Angiopteris type or the derived Adiantum type via a single inversion; (ii the trnY-trnE intergenic spacer (IGS of the filmy fern Vandenboschia radicans was expanded 3-fold due to the tandem 27-bp repeats which showed strong sequence similarity with the anticodon domain of trnY; (iii the trnY-trnE IGSs of two horsetail ferns Equisetum ramosissimum and E. arvense underwent an unprecedented 5-kb long expansion, more than a quarter of which was consisted of a single type of direct repeats also relevant to the trnY anticodon domain; and (iv ycf66 has independently lost at least four times in ferns. Conclusions Our results provided fresh insights into the evolutionary process of fern BZ regions. The intermediate BZ gene order was not detected, supporting that the Adiantum type was generated by two inversions occurring in pairs. The occurrence of Vandenboschia 27-bp repeats represents the first evidence of partial tRNA gene duplication in fern plastomes. Repeats potentially forming a stem-loop structure play major roles in the expansion of the trnY-trnE IGS.

  4. Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

    Science.gov (United States)

    Sharifi Tabar, Mehdi; Habashi, Ali Akbar; Rajabi Memari, Hamid

    2013-01-01

    Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

  5. Phylogeny, character evolution, and biogeography of Cuscuta (dodders; Convolvulaceae) inferred from coding plastid and nuclear sequences.

    Science.gov (United States)

    García, Miguel A; Costea, Mihai; Kuzmina, Maria; Stefanović, Saša

    2014-04-01

    The parasitic genus Cuscuta, containing some 200 species circumscribed traditionally in three subgenera, is nearly cosmopolitan, occurring in a wide range of habitats and hosts. Previous molecular studies, on subgenera Grammica and Cuscuta, delimited major clades within these groups. However, the sequences used were unalignable among subgenera, preventing the phylogenetic comparison across the genus. We conducted a broad phylogenetic study using rbcL and nrLSU sequences covering the morphological, physiological, and geographical diversity of Cuscuta. We used parsimony methods to reconstruct ancestral states for taxonomically important characters. Biogeographical inferences were obtained using statistical and Bayesian approaches. Four well-supported major clades are resolved. Two of them correspond to subgenera Monogynella and Grammica. Subgenus Cuscuta is paraphyletic, with section Pachystigma sister to subgenus Grammica. Previously described cases of strongly supported discordance between plastid and nuclear phylogenies, interpreted as reticulation events, are confirmed here and three new cases are detected. Dehiscent fruits and globose stigmas are inferred as ancestral character states, whereas the ancestral style number is ambiguous. Biogeographical reconstructions suggest an Old World origin for the genus and subsequent spread to the Americas as a consequence of one long-distance dispersal. Hybridization may play an important yet underestimated role in the evolution of Cuscuta. Our results disagree with scenarios of evolution (polarity) previously proposed for several taxonomically important morphological characters, and with their usage and significance. While several cases of long-distance dispersal are inferred, vicariance or dispersal to adjacent areas emerges as the dominant biogeographical pattern.

  6. A gene phylogeny of the red algae (Rhodophyta) based on plastid rbcL.

    Science.gov (United States)

    Freshwater, D W; Fredericq, S; Butler, B S; Hommersand, M H; Chase, M W

    1994-01-01

    A phylogeny for the Rhodophyta has been inferred by parsimony analysis of plastid rbcL sequences representing 81 species, 68 genera, 38 families, and 17 orders of red algae; rbcL encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Levels of sequence divergence among species, genera, and families are high in red algae, typically much greater than those reported for flowering plants. The Rhodophyta traditionally consists of one class, Rhodophyceae, and two subclasses, Bangiophycidae and Florideophycidae. The Bangiophycidae with three orders (Porphyridiales, Compsopogonales, and Bangiales) appears to be polyphyletic, and the Florideophycidae with 17 orders is monophyletic in this study. The current classification of the Florideophycidae based on ultrastructure of pit connections is supported. With the exception of the Rhodogorgonales, which appears to be misplaced, orders with one or two pit-plug cap layers (Hildenbrandiales, Corallinales, Acrochaetiales, Palmanales, Batrachospermales, and Nemaliales) terminate long branches of basal position within Florideophycidae in the most parsimonious rbcL tree. Orders that lack typical cap layers but possess a cap membrane are resolved as a monophyletic clade sister to the Ahnfeltiales. The large order Gigartinales, which is distributed among five rbcL clades, is polyphyletic. Families that possess typical carrageenan in their cell walls are resolved as a terminal clade containing two family complexes centered around the Solieriaceae and Gigartinaceae. PMID:8041781

  7. Phylogeny of Celastrus L. (Celastraceae) inferred from two nuclear and three plastid markers.

    Science.gov (United States)

    Mu, Xian-Yun; Zhao, Liang-Cheng; Zhang, Zhi-Xiang

    2012-09-01

    This is the first comprehensive molecular investigation of the genus Celastrus L. Phylogenetic relationships within the genus were assessed based on sequences of two nuclear (ETS, ITS) and three plastid (psbA-trnH, rpl16 and trnL-F) regions using the Bayesian inference and the maximum parsimony methods. Our results show that Celastrus, together with Tripterygium, formed a maximal supported clade. Within the cluster, Celastrus is composed of a basal clade and a core Celastrus clade, and the latter is consisted of six subclades. Relationships among species are more influenced by latitude than continental distribution patterns. The cauline cyme and lunate seeds are distinct characters to one of the maximal supported subclades. Their close relationship, similar geographical pattern and habitat imply that C. flagellaris may be a potential invasive species threatening C. scandens in North America. Celastrus leiocarpus, C. oblanceifolius and C. rugosus are confirmed as synonyms of C. punctatus, C. aculeatus and C. glaucophyllus, respectively. Discordance between the molecular data and previous morphology-based subgeneric classifications are noted. More works are needed to clarify the relationship between Celastrus and Tripterygium and the species within Celastrus.

  8. A phylogenetic analysis of Diurideae (Orchidaceae) based on plastid DNA sequence data.

    Science.gov (United States)

    Kores, P J; Molvray, M; Weston, P H; Hopper, S D; Brown, A P; Cameron, K M; Chase, M W

    2001-10-01

    DNA sequence data from plastid matK and trnL-F regions were used in phylogenetic analyses of Diurideae, which indicate that Diurideae are not monophyletic as currently delimited. However, if Chloraeinae and Pterostylidinae are excluded from Diurideae, the remaining subtribes form a well-supported, monophyletic group that is sister to a "spiranthid" clade. Chloraea, Gavilea, and Megastylis pro parte (Chloraeinae) are all placed among the spiranthid orchids and form a grade with Pterostylis leading to a monophyletic Cranichideae. Codonorchis, previously included among Chloraeinae, is sister to Orchideae. Within the more narrowly delimited Diurideae two major lineages are apparent. One includes Diuridinae, Cryptostylidinae, Thelymitrinae, and an expanded Drakaeinae; the other includes Caladeniinae s.s., Prasophyllinae, and Acianthinae. The achlorophyllous subtribe Rhizanthellinae is a member of Diurideae, but its placement is otherwise uncertain. The sequence-based trees indicate that some morphological characters used in previous classifications, such as subterranean storage organs, anther position, growth habit, fungal symbionts, and pollination syndromes have more complex evolutionary histories than previously hypothesized. Treatments based upon these characters have produced conflicting classifications, and molecular data offer a tool for reevaluating these phylogenetic hypotheses.

  9. Phylogenetic relationships in Peniocereus (Cactaceae) inferred from plastid DNA sequence data.

    Science.gov (United States)

    Arias, Salvador; Terrazas, Teresa; Arreola-Nava, Hilda J; Vázquez-Sánchez, Monserrat; Cameron, Kenneth M

    2005-10-01

    The phylogenetic relationships of Peniocereus (Cactaceae) species were studied using parsimony analyses of DNA sequence data. The plastid rpl16 and trnL-F regions were sequenced for 98 taxa including 17 species of Peniocereus, representatives from all genera of tribe Pachycereeae, four genera of tribe Hylocereeae, as well as from three additional outgroup genera of tribes Calymmantheae, Notocacteae, and Trichocereeae. Phylogenetic analyses support neither the monophyly of Peniocereus as currently circumscribed, nor the monophyly of tribe Pachycereeae since species of Peniocereus subgenus Pseudoacanthocereus are embedded within tribe Hylocereeae. Furthermore, these results show that the eight species of Peniocereus subgenus Peniocereus (Peniocereus sensu stricto) form a well-supported clade within subtribe Pachycereinae; P. serpentinus is also a member of this subtribe, but is sister to Bergerocactus. Moreover, Nyctocereus should be resurrected as a monotypic genus. Species of Peniocereus subgenus Pseudoacanthocereus are positioned among species of Acanthocereus within tribe Hylocereeae, indicating that they may be better classified within that genus. A number of morphological and anatomical characters, especially related to the presence or absence of dimorphic branches, are discussed to support these relationships.

  10. Nuclear-cytoplasmic conflict in pea (Pisum sativum L. is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

    Directory of Open Access Journals (Sweden)

    Vera S Bogdanova

    Full Text Available In crosses of wild and cultivated peas (Pisum sativum L., nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

  11. Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

    Science.gov (United States)

    Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

    2015-01-01

    In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

  12. Morphological and quantitative changes in mitochondria, plastids, and peroxisomes during the log-to-stationary transition of the growth phase in cultured tobacco BY-2 cells.

    Science.gov (United States)

    Toyooka, Kiminori; Sato, Mayuko; Wakazaki, Mayumi; Matsuoka, Ken

    2016-01-01

    We developed a wide-range and high-resolution transmission electron microscope acquisition system and obtained giga-pixel images of tobacco BY-2 cells during the log and stationary phases of cell growth. We demonstrated that the distribution and ultrastructure of compartments involved in membrane traffic (i.e., Golgi apparatus, multivesicular body, and vesicle cluster) change during the log-to-stationary transition. Mitochondria, peroxisomes, and plastids were also enumerated. Electron densities of mitochondria and peroxisomes were altered during the growth-phase shift, while their numbers were reduced by nearly half. Plastid structure dramatically changed from atypical to spherical with starch granules. Nearly the same number of plastids was observed in both log and stationary phases. These results indicate that mechanisms regulating organelle populations differ from organelle to organelle.

  13. Recombination-dependent replication and gene conversion homogenize repeat sequences and diversify plastid genome structure.

    Science.gov (United States)

    Ruhlman, Tracey A; Zhang, Jin; Blazier, John C; Sabir, Jamal S M; Jansen, Robert K

    2017-04-01

    There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements. We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements. Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent ∼22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements. We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats. © 2017 Botanical Society of America.

  14. Organization of plastid genomes in the freshwater red algal order Batrachospermales (Rhodophyta).

    Science.gov (United States)

    Paiano, Monica Orlandi; Del Cortona, Andrea; Costa, Joana F; Liu, Shao-Lun; Verbruggen, Heroen; De Clerck, Olivier; Necchi, Orlando

    2018-02-01

    Little is known about genome organization in members of the order Batrachospermales, and the infra-ordinal relationship remains unresolved. Plastid (cp) genomes of seven members of the freshwater red algal order Batrachospermales were sequenced, with the following aims: (i) to describe the characteristics of cp genomes and compare these with other red algal groups; (ii) to infer the phylogenetic relationships among these members to better understand the infra-ordinal classification. Cp genomes of Batrachospermales are large, with several cases of gene loss, they are gene-dense (high gene content for the genome size and short intergenic regions) and have highly conserved gene order. Phylogenetic analyses based on concatenated nucleotide genome data roughly supports the current taxonomic system for the order. Comparative analyses confirm data for members of the class Florideophyceae that cp genomes in Batrachospermales is highly conserved, with little variation in gene composition. However, relevant new features were revealed in our study: genome sizes in members of Batrachospermales are close to the lowest values reported for Florideophyceae; differences in cp genome size within the order are large in comparison with other orders (Ceramiales, Gelidiales, Gracilariales, Hildenbrandiales, and Nemaliales); and members of Batrachospermales have the lowest number of protein-coding genes among the Florideophyceae. In terms of gene loss, apcF, which encodes the allophycocyanin beta subunit, is absent in all sequenced taxa of Batrachospermales. We reinforce that the interordinal relationships between the freshwater orders Batrachospermales and Thoreales within the Nemaliophycidae is not well resolved due to limited taxon sampling. © 2017 Phycological Society of America.

  15. Phylogeny of Celastraceae tribe Euonymeae inferred from morphological characters and nuclear and plastid genes.

    Science.gov (United States)

    Simmons, Mark P; McKenna, Miles J; Bacon, Christine D; Yakobson, Kendra; Cappa, Jennifer J; Archer, Robert H; Ford, Andrew J

    2012-01-01

    The phylogeny of Celastraceae tribe Euonymeae (≈ 230 species in eight genera in both the Old and New Worlds) was inferred using morphological characters together with plastid (matK, trnL-F) and nuclear (ITS and 26S rDNA) genes. Tribe Euonymeae has been defined as those genera of Celastraceae with generally opposite leaves, isomerous carpels, loculicidally dehiscent capsules, and arillate seeds (except Microtropis). Euonymus is the most diverse (129 species) and widely cultivated genus in the tribe. We infer that tribe Euonymeae consists of at least six separate lineages within Celastraceae and that a revised natural classification of the family is needed. Microtropis and Quetzalia are inferred to be distinct sister groups that together are sister to Zinowiewia. The endangered Monimopetalum chinense is an isolated and early derived lineage of Celastraceae that represents an important component of phylogenetic diversity within the family. Hedraianthera is sister to Brassiantha, and we describe a second species (Brassiantha hedraiantheroides A.J. Ford) that represents the first reported occurrence of this genus in Australia. Euonymus globularis, from eastern Australia, is sister to Menepetalum, which is endemic to New Caledonia, and we erect a new genus (Dinghoua R.H. Archer) for it. The Madagascan species of Euonymus are sister to Pleurostylia and recognized as a distinct genus (Astrocassine ined.). Glyptopetalum, Torralbasia, and Xylonymus are all closely related to Euonymus sensu stricto and are questionably distinct from it. Current intrageneric classifications of Euonymus are not completely natural and require revision. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Phylogeny of Celastraceae subfamily Hippocrateoideae inferred from morphological characters and nuclear and plastid loci.

    Science.gov (United States)

    Coughenour, Jennifer M; Simmons, Mark P; Lombardi, Julio A; Yakobson, Kendra; Archer, Robert H

    2011-05-01

    The phylogeny of Celastraceae subfamily Hippocrateoideae (∼ 100 species and 19 genera in the Old and New World tropics) was inferred using morphological characters together with plastid (matK, trnL-F) and nuclear (ITS and 26S rDNA) genes. The subfamily is easily recognized by the synapomorphies of transversely flattened, deeply lobed capsules and seeds with membranous basal wings or narrow stipes together with bisexual, 5-merous flowers that generally have an extrastaminal disk and three stamens. Hippocrateoideae, like Salacioideae, are inferred to have an Old World origin. The narrow stipes of Neotropical species that are water-dispersed are inferred to be derived within the subfamily from ancestral species with wind-dispersed winged seeds. Helictonema, a monotypic genus endemic to tropical Africa, has a small, white, spongy aril that is located at the base of the seed wing and appears to be unique within Hippocrateoideae. Our inference that Helictonema is sister to the remaining members of the subfamily, considered in the context of Sarawakodendron being sister to Salacioideae, suggests that small arils and capsular fruit were primitive within both subfamilies. The aril became dramatically enlarged within Salacioideae, in which the fruits are berries, and lost entirely within Hippocrateoideae, in which the fruits are transversely flattened capsules. All five Old World taxa of Prionostemma and all eight currently recognized species within Simirestis are transferred to Pristimera, one South African variety of Pristimera is raised to species level, and all three taxa in Pristimera subgenus Trochantha are transferred to the new genus Trochantha. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

    Directory of Open Access Journals (Sweden)

    Hongyu Chen

    2018-02-01

    Full Text Available In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem, cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

  18. Plastid phylogenomics and adaptive evolution of Gaultheria series Trichophyllae (Ericaceae), a clade from sky islands of the Himalaya-Hengduan Mountains.

    Science.gov (United States)

    Zhang, Ming-Ying; Fritsch, Peter W; Ma, Peng-Fei; Wang, Hong; Lu, Lu; Li, De-Zhu

    2017-05-01

    Gaultheria series Trichophyllae Airy Shaw is an angiosperm clade of high-alpine shrublets endemic to the Himalaya-Hengduan Mountains and characterized by recent species divergence and convergent character evolution that has until recently caused much confusion in species circumscription. Although multiple DNA sequence regions have been employed previously, phylogenetic relationships among species in the group have remained largely unresolved. Here we examined the effectiveness of the plastid genome for improving phylogenetic resolution within the G. series Trichophyllae clade. Plastid genomes of 31 samples representing all 19 recognized species of the series and three outgroup species were sequenced with Illumina Sequencing technology. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) phylogenetic analyses were performed with various datasets, i.e., that from the whole plastid genome, coding regions, noncoding regions, large single-copy region (LSC) and inverted-repeat region a (IRa). The partitioned whole plastid genome with inverted-repeat region b (IRb) excluded was also analyzed with ML and BI. Tree topologies based on the whole plastid genome, noncoding regions, and LSC region datasets across all analyses, and that based on the partitioned dataset with ML and BI analyses, are identical and generally strongly supported. Gaultheria series Trichophyllae form a clade with three species and one variety that is sister to a clade of the remaining 16 species; the latter comprises seven main subclades. Interspecific relationships within the series are strongly supported except for those based on the coding-region and IRa-region datasets. Eight divergence hotspot regions, each possessing >5% percent variable sites, were screened across the whole plastid genome of the 28 individuals sampled in the series. Results of morphological character evolution reconstruction diagnose several clades, and a hypothesis of adaptive evolution for plant habit is

  19. The plastid genome of Najas flexilis: adaptation to submersed environments is accompanied by the complete loss of the NDH complex in an aquatic angiosperm.

    Directory of Open Access Journals (Sweden)

    Elena L Peredo

    Full Text Available The re-colonization of aquatic habitats by angiosperms has presented a difficult challenge to plants whose long evolutionary history primarily reflects adaptations to terrestrial conditions. Many aquatics must complete vital stages of their life cycle on the water surface by means of floating or emergent leaves and flowers. Only a few species, mainly within the order Alismatales, are able to complete all aspects of their life cycle including pollination, entirely underwater. Water-pollinated Alismatales include seagrasses and water nymphs (Najas, the latter being the only freshwater genus in the family Hydrocharitaceae with subsurface water-pollination. We have determined the complete nucleotide sequence of the plastid genome of Najas flexilis. The plastid genome of N. flexilis is a circular AT-rich DNA molecule of 156 kb, which displays a quadripartite structure with two inverted repeats (IR separating the large single copy (LSC from the small single copy (SSC regions. In N. flexilis, as in other Alismatales, the rps19 and trnH genes are localized in the LSC region instead of within the IR regions as in other monocots. However, the N. flexilis plastid genome presents some anomalous modifications. The size of the SSC region is only one third of that reported for closely related species. The number of genes in the plastid is considerably less. Both features are due to loss of the eleven ndh genes in the Najas flexilis plastid. In angiosperms, the absence of ndh genes has been related mainly to the loss of photosynthetic function in parasitic plants. The ndh genes encode the NAD(PH dehydrogenase complex, believed essential in terrestrial environments, where it increases photosynthetic efficiency in variable light intensities. The modified structure of the N. flexilis plastid genome suggests that adaptation to submersed environments, where light is scarce, has involved the loss of the NDH complex in at least some photosynthetic angiosperms.

  20. DNA Barcoding the Canadian Arctic Flora: Core Plastid Barcodes (rbcL + matK) for 490 Vascular Plant Species

    Science.gov (United States)

    Saarela, Jeffery M.; Sokoloff, Paul C.; Gillespie, Lynn J.; Consaul, Laurie L.; Bull, Roger D.

    2013-01-01

    Accurate identification of Arctic plant species is critical for understanding potential climate-induced changes in their diversity and distributions. To facilitate rapid identification we generated DNA barcodes for the core plastid barcode loci (rbcL and matK) for 490 vascular plant species, representing nearly half of the Canadian Arctic flora and 93% of the flora of the Canadian Arctic Archipelago. Sequence recovery was higher for rbcL than matK (93% and 81%), and rbcL was easier to recover than matK from herbarium specimens (92% and 77%). Distance-based and sequence-similarity analyses of combined rbcL + matK data discriminate 97% of genera, 56% of species, and 7% of infraspecific taxa. There is a significant negative correlation between the number of species sampled per genus and the percent species resolution per genus. We characterize barcode variation in detail in the ten largest genera sampled (Carex, Draba, Festuca, Pedicularis, Poa, Potentilla, Puccinellia, Ranunculus, Salix, and Saxifraga) in the context of their phylogenetic relationships and taxonomy. Discrimination with the core barcode loci in these genera ranges from 0% in Salix to 85% in Carex. Haplotype variation in multiple genera does not correspond to species boundaries, including Taraxacum, in which the distribution of plastid haplotypes among Arctic species is consistent with plastid variation documented in non-Arctic species. Introgression of Poa glauca plastid DNA into multiple individuals of P. hartzii is problematic for identification of these species with DNA barcodes. Of three supplementary barcode loci (psbA–trnH, psbK–psbI, atpF–atpH) collected for a subset of Poa and Puccinellia species, only atpF–atpH improved discrimination in Puccinellia, compared with rbcL and matK. Variation in matK in Vaccinium uliginosum and rbcL in Saxifraga oppositifolia corresponds to variation in other loci used to characterize the phylogeographic histories of these Arctic-alpine species. PMID

  1. Molecular phylogeny of Edraianthus (Grassy Bells; Campanulaceae) based on non-coding plastid DNA sequences

    DEFF Research Database (Denmark)

    Stefanovic, Sasa; Lakusic, Dmitar; Kuzmina, Maria

    2008-01-01

    The Balkan Peninsula is known as an ice-age refugium and an area with high rates of speciation and diversification. Only a few genera have their centers of distribution in the Balkans and the endemic genus Edraianthus is one of its most prominent groups. As such, Edraianthus is an excellent model...... divided into three sections: E. sect. Edraianthus, E. sect. Uniflori, and E. sect. Spathulati. We present here the first phylogenetic study of Edraianthus based on multiple plastid DNA sequences (trnL-F region and rbcL-atpB spacer) derived from a wide taxonomic sampling and geographic range. While...

  2. Plastome Evolution in the Sole Hemiparasitic Genus Laurel Dodder (Cassytha) and Insights into the Plastid Phylogenomics of Lauraceae.

    Science.gov (United States)

    Wu, Chung-Shien; Wang, Ting-Jen; Wu, Chia-Wen; Wang, Ya-Nan; Chaw, Shu-Miaw

    2017-10-01

    To date, little is known about the evolution of plastid genomes (plastomes) in Lauraceae. As one of the top five largest families in tropical forests, the Lauraceae contain many species that are important ecologically and economically. Lauraceous species also provide wonderful materials to study the evolutionary trajectory in response to parasitism because they contain both nonparasitic and parasitic species. This study compared the plastomes of nine Lauraceous species, including the sole hemiparasitic and herbaceous genus Cassytha (laurel dodder; here represented by Cassytha filiformis). We found differential contractions of the canonical inverted repeat (IR), resulting in two IR types present in Lauraceae. These two IR types reinforce Cryptocaryeae and Neocinnamomum-Perseeae-Laureae as two separate clades. Our data reveal several traits unique to Cas. filiformis, including loss of IRs, loss or pseudogenization of 11 ndh and rpl23 genes, richness of repeats, and accelerated rates of nucleotide substitutions in protein-coding genes. Although Cas. filiformis is low in chlorophyll content, our analysis based on dN/dS ratios suggests that both its plastid house-keeping and photosynthetic genes are under strong selective constraints. Hence, we propose that short generation time and herbaceous lifestyle rather than reduced photosynthetic ability drive the accelerated rates of nucleotide substitutions in Cas. filiformis. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  4. Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae.

    Science.gov (United States)

    Wei, Li; Xin, Yi; Wang, Dongmei; Jing, Xiaoyan; Zhou, Qian; Su, Xiaoquan; Jia, Jing; Ning, Kang; Chen, Feng; Hu, Qiang; Xu, Jian

    2013-08-05

    Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes. Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species. This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of

  5. Isoprenoid, lipid, and protein contents in intact plastids isolated from mesocarp cells of traditional and high-pigment tomato cultivars at different ripening stages.

    Science.gov (United States)

    Lenucci, Marcello S; Serrone, Lucia; De Caroli, Monica; Fraser, Paul D; Bramley, Peter M; Piro, Gabriella; Dalessandro, Giuseppe

    2012-02-22

    This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, β-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 μg/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 μg/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and α-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of ~41.6 kDa, was found to increase in intensity.

  6. The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. sequence evaluation and plastome evolution.

    Science.gov (United States)

    Greiner, Stephan; Wang, Xi; Rauwolf, Uwe; Silber, Martina V; Mayer, Klaus; Meurer, Jörg; Haberer, Georg; Herrmann, Reinhold G

    2008-04-01

    The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome-genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I-III in one clade, while plastome IV appears to be closest to the common ancestor.

  7. The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. Sequence evaluation and plastome evolution†

    Science.gov (United States)

    Greiner, Stephan; Wang, Xi; Rauwolf, Uwe; Silber, Martina V.; Mayer, Klaus; Meurer, Jörg; Haberer, Georg; Herrmann, Reinhold G.

    2008-01-01

    The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome–genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common ancestor. PMID:18299283

  8. Phylogeny reconstruction and hybrid analysis of populus (Salicaceae) based on nucleotide sequences of multiple single-copy nuclear genes and plastid fragments.

    Science.gov (United States)

    Wang, Zhaoshan; Du, Shuhui; Dayanandan, Selvadurai; Wang, Dongsheng; Zeng, Yanfei; Zhang, Jianguo

    2014-01-01

    Populus (Salicaceae) is one of the most economically and ecologically important genera of forest trees. The complex reticulate evolution and lack of highly variable orthologous single-copy DNA markers have posed difficulties in resolving the phylogeny of this genus. Based on a large data set of nuclear and plastid DNA sequences, we reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed better resolution at both inter- and intra-sectional level than previous studies. The results revealed that (1) the plastid-based phylogenetic tree resulted in two main clades, suggesting an early divergence of the maternal progenitors of Populus; (2) three advanced sections (Populus, Aigeiros and Tacamahaca) are of hybrid origin; (3) species of the section Tacamahaca could be divided into two major groups based on plastid and nuclear DNA data, suggesting a polyphyletic nature of the section; and (4) many species proved to be of hybrid origin based on the incongruence between plastid and nuclear DNA trees. Reticulate evolution may have played a significant role in the evolution history of Populus by facilitating rapid adaptive radiations into different environments.

  9. Phylogeny reconstruction and hybrid analysis of populus (Salicaceae based on nucleotide sequences of multiple single-copy nuclear genes and plastid fragments.

    Directory of Open Access Journals (Sweden)

    Zhaoshan Wang

    Full Text Available Populus (Salicaceae is one of the most economically and ecologically important genera of forest trees. The complex reticulate evolution and lack of highly variable orthologous single-copy DNA markers have posed difficulties in resolving the phylogeny of this genus. Based on a large data set of nuclear and plastid DNA sequences, we reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed better resolution at both inter- and intra-sectional level than previous studies. The results revealed that (1 the plastid-based phylogenetic tree resulted in two main clades, suggesting an early divergence of the maternal progenitors of Populus; (2 three advanced sections (Populus, Aigeiros and Tacamahaca are of hybrid origin; (3 species of the section Tacamahaca could be divided into two major groups based on plastid and nuclear DNA data, suggesting a polyphyletic nature of the section; and (4 many species proved to be of hybrid origin based on the incongruence between plastid and nuclear DNA trees. Reticulate evolution may have played a significant role in the evolution history of Populus by facilitating rapid adaptive radiations into different environments.

  10. Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Tadini, Luca; Ferrari, Roberto; Lehniger, Marie-Kristin; Mizzotti, Chiara; Moratti, Fabio; Resentini, Francesca; Colombo, Monica; Costa, Alex; Masiero, Simona; Pesaresi, Paolo

    2018-04-23

    AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

  11. Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

    Science.gov (United States)

    Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

    2011-01-01

    Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

  12. Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds.

    Science.gov (United States)

    Tsuji, Yoshinori; Suzuki, Iwane; Shiraiwa, Yoshihiro

    2012-06-01

    Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.

  13. Nuclear and plastid haplotypes suggest rapid diploid and polyploid speciation in the N Hemisphere Achillea millefolium complex (Asteraceae

    Directory of Open Access Journals (Sweden)

    Guo Yan-Ping

    2012-01-01

    Full Text Available Abstract Background Species complexes or aggregates consist of a set of closely related species often of different ploidy levels, whose relationships are difficult to reconstruct. The N Hemisphere Achillea millefolium aggregate exhibits complex morphological and genetic variation and a broad ecological amplitude. To understand its evolutionary history, we study sequence variation at two nuclear genes and three plastid loci across the natural distribution of this species complex and compare the patterns of such variations to the species tree inferred earlier from AFLP data. Results Among the diploid species of A. millefolium agg., gene trees of the two nuclear loci, ncpGS and SBP, and the combined plastid fragments are incongruent with each other and with the AFLP tree likely due to incomplete lineage sorting or secondary introgression. In spite of the large distributional range, no isolation by distance is found. Furthermore, there is evidence for intragenic recombination in the ncpGS gene. An analysis using a probabilistic model for population demographic history indicates large ancestral effective population sizes and short intervals between speciation events. Such a scenario explains the incongruence of the gene trees and species tree we observe. The relationships are particularly complex in the polyploid members of A. millefolium agg. Conclusions The present study indicates that the diploid members of A. millefolium agg. share a large part of their molecular genetic variation. The findings of little lineage sorting and lack of isolation by distance is likely due to short intervals between speciation events and close proximity of ancestral populations. While previous AFLP data provide species trees congruent with earlier morphological classification and phylogeographic considerations, the present sequence data are not suited to recover the relationships of diploid species in A. millefolium agg. For the polyploid taxa many hybrid links and

  14. Negative correlation between rates of molecular evolution and flowering cycles in temperate woody bamboos revealed by plastid phylogenomics.

    Science.gov (United States)

    Ma, Peng-Fei; Vorontsova, Maria S; Nanjarisoa, Olinirina Prisca; Razanatsoa, Jacqueline; Guo, Zhen-Hua; Haevermans, Thomas; Li, De-Zhu

    2017-12-21

    Heterogeneous rates of molecular evolution are universal across the tree of life, posing challenges for phylogenetic inference. The temperate woody bamboos (tribe Arundinarieae, Poaceae) are noted for their extremely slow molecular evolutionary rates, supposedly caused by their mysterious monocarpic reproduction. However, the correlation between substitution rates and flowering cycles has not been formally tested. Here we present 15 newly sequenced plastid genomes of temperate woody bamboos, including the first genomes ever sequenced from Madagascar representatives. A data matrix of 46 plastid genomes representing all 12 lineages of Arundinarieae was assembled for phylogenetic and molecular evolutionary analyses. We conducted phylogenetic analyses using different sequences (e.g., coding and noncoding) combined with different data partitioning schemes, revealing conflicting relationships involving internodes among several lineages. A great difference in branch lengths were observed among the major lineages, and topological inconsistency could be attributed to long-branch attraction (LBA). Using clock model-fitting by maximum likelihood and Bayesian approaches, we furthermore demonstrated extensive rate variation among these major lineages. Rate accelerations mainly occurred for the isolated lineages with limited species diversification, totaling 11 rate shifts during the tribe's evolution. Using linear regression analysis, we found a negative correlation between rates of molecular evolution and flowering cycles for Arundinarieae, notwithstanding that the correlation maybe insignificant when taking the phylogenetic structure into account. Using the temperate woody bamboos as an example, we found further evidence that rate heterogeneity is universal in plants, suggesting that this will pose a challenge for phylogenetic reconstruction of bamboos. The bamboos with longer flowering cycles tend to evolve more slowly than those with shorter flowering cycles, in accordance

  15. Arabidopsis plastid AMOS1/EGY1 integrates abscisic acid signaling to regulate global gene expression response to ammonium stress

    KAUST Repository

    Li, Baohai

    2012-10-12

    Ammonium (NH4 +) is a ubiquitous intermediate of nitrogen metabolism but is notorious for its toxic effects on most organisms. Extensive studies of the underlying mechanisms of NH4 + toxicity have been reported in plants, but it is poorly understood how plants acclimate to high levels of NH4 +. Here, we identified an Arabidopsis (Arabidopsis thaliana) mutant, ammonium overly sensitive1 (amos1), that displays severe chlorosis under NH4 + stress. Map-based cloning shows amos1 to carry a mutation in EGY1 (for ethylene-dependent, gravitropism-deficient, and yellow-green-like protein1), which encodes a plastid metalloprotease. Transcriptomic analysis reveals that among the genes activated in response to NH4 +, 90% are regulated dependent on AMOS1/ EGY1. Furthermore, 63% of AMOS1/EGY1-dependent NH4 +-activated genes contain an ACGTG motif in their promoter region, a core motif of abscisic acid (ABA)-responsive elements. Consistent with this, our physiological, pharmacological, transcriptomic, and genetic data show that ABA signaling is a critical, but not the sole, downstream component of the AMOS1/EGY1-dependent pathway that regulates the expression of NH4 +-responsive genes and maintains chloroplast functionality under NH4 + stress. Importantly, abi4 mutants defective in ABA-dependent and retrograde signaling, but not ABA-deficient mutants, mimic leaf NH4 + hypersensitivity of amos1. In summary, our findings suggest that an NH4 +-responsive plastid retrograde pathway, which depends on AMOS1/EGY1 function and integrates with ABA signaling, is required for the regulation of expression of the presence of high NH4 + levels. © 2012 American Society of Plant Biologists. All Rights Reserved.

  16. Arabidopsis plastid AMOS1/EGY1 integrates abscisic acid signaling to regulate global gene expression response to ammonium stress

    KAUST Repository

    Li, Baohai; Li, Qing; Xiong, Liming; Kronzucker, Herbert J.; Krä mer, Ute; Shi, Weiming

    2012-01-01

    Ammonium (NH4 +) is a ubiquitous intermediate of nitrogen metabolism but is notorious for its toxic effects on most organisms. Extensive studies of the underlying mechanisms of NH4 + toxicity have been reported in plants, but it is poorly understood how plants acclimate to high levels of NH4 +. Here, we identified an Arabidopsis (Arabidopsis thaliana) mutant, ammonium overly sensitive1 (amos1), that displays severe chlorosis under NH4 + stress. Map-based cloning shows amos1 to carry a mutation in EGY1 (for ethylene-dependent, gravitropism-deficient, and yellow-green-like protein1), which encodes a plastid metalloprotease. Transcriptomic analysis reveals that among the genes activated in response to NH4 +, 90% are regulated dependent on AMOS1/ EGY1. Furthermore, 63% of AMOS1/EGY1-dependent NH4 +-activated genes contain an ACGTG motif in their promoter region, a core motif of abscisic acid (ABA)-responsive elements. Consistent with this, our physiological, pharmacological, transcriptomic, and genetic data show that ABA signaling is a critical, but not the sole, downstream component of the AMOS1/EGY1-dependent pathway that regulates the expression of NH4 +-responsive genes and maintains chloroplast functionality under NH4 + stress. Importantly, abi4 mutants defective in ABA-dependent and retrograde signaling, but not ABA-deficient mutants, mimic leaf NH4 + hypersensitivity of amos1. In summary, our findings suggest that an NH4 +-responsive plastid retrograde pathway, which depends on AMOS1/EGY1 function and integrates with ABA signaling, is required for the regulation of expression of the presence of high NH4 + levels. © 2012 American Society of Plant Biologists. All Rights Reserved.

  17. Comparative analyses of plastid sequences between native and introduced populations of aquatic weeds Elodea canadensis and E. nuttallii.

    Directory of Open Access Journals (Sweden)

    Tea Huotari

    Full Text Available Non-indigenous species (NIS are species living outside their historic or native range. Invasive NIS often cause severe environmental impacts, and may have large economical and social consequences. Elodea (Hydrocharitaceae is a New World genus with at least five submerged aquatic angiosperm species living in fresh water environments. Our aim was to survey the geographical distribution of cpDNA haplotypes within the native and introduced ranges of invasive aquatic weeds Elodea canadensis and E. nuttallii and to reconstruct the spreading histories of these invasive species. In order to reveal informative chloroplast (cp genome regions for phylogeographic analyses, we compared the plastid sequences of native and introduced individuals of E. canadensis. In total, we found 235 variable sites (186 SNPs, 47 indels and two inversions between the two plastid sequences consisting of 112,193 bp and developed primers flanking the most variable genomic areas. These 29 primer pairs were used to compare the level and pattern of intraspecific variation within E. canadensis to interspecific variation between E. canadensis and E. nuttallii. Nine potentially informative primer pairs were used to analyze the phylogeographic structure of both Elodea species, based on 70 E. canadensis and 25 E. nuttallii individuals covering native and introduced distributions. On the whole, the level of variation between the two Elodea species was 53% higher than that within E. canadensis. In our phylogeographic analysis, only a single haplotype was found in the introduced range in both species. These haplotypes H1 (E. canadensis and A (E. nuttallii were also widespread in the native range, covering the majority of native populations analyzed. Therefore, we were not able to identify either the geographic origin of the introduced populations or test the hypothesis of single versus multiple introductions. The divergence between E. canadensis haplotypes was surprisingly high, and future

  18. Comparative analyses of plastid sequences between native and introduced populations of aquatic weeds Elodea canadensis and E. nuttallii.

    Science.gov (United States)

    Huotari, Tea; Korpelainen, Helena

    2013-01-01

    Non-indigenous species (NIS) are species living outside their historic or native range. Invasive NIS often cause severe environmental impacts, and may have large economical and social consequences. Elodea (Hydrocharitaceae) is a New World genus with at least five submerged aquatic angiosperm species living in fresh water environments. Our aim was to survey the geographical distribution of cpDNA haplotypes within the native and introduced ranges of invasive aquatic weeds Elodea canadensis and E. nuttallii and to reconstruct the spreading histories of these invasive species. In order to reveal informative chloroplast (cp) genome regions for phylogeographic analyses, we compared the plastid sequences of native and introduced individuals of E. canadensis. In total, we found 235 variable sites (186 SNPs, 47 indels and two inversions) between the two plastid sequences consisting of 112,193 bp and developed primers flanking the most variable genomic areas. These 29 primer pairs were used to compare the level and pattern of intraspecific variation within E. canadensis to interspecific variation between E. canadensis and E. nuttallii. Nine potentially informative primer pairs were used to analyze the phylogeographic structure of both Elodea species, based on 70 E. canadensis and 25 E. nuttallii individuals covering native and introduced distributions. On the whole, the level of variation between the two Elodea species was 53% higher than that within E. canadensis. In our phylogeographic analysis, only a single haplotype was found in the introduced range in both species. These haplotypes H1 (E. canadensis) and A (E. nuttallii) were also widespread in the native range, covering the majority of native populations analyzed. Therefore, we were not able to identify either the geographic origin of the introduced populations or test the hypothesis of single versus multiple introductions. The divergence between E. canadensis haplotypes was surprisingly high, and future research may

  19. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    International Nuclear Information System (INIS)

    Torreira, Eva; Seabra, Ana Rita; Marriott, Hazel; Zhou, Min; Llorca, Óscar; Robinson, Carol V.; Carvalho, Helena G.; Fernández-Tornero, Carlos; Pereira, Pedro José Barbosa

    2014-01-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants

  20. Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance.

    Science.gov (United States)

    Kumar, Shashi; Dhingra, Amit; Daniell, Henry

    2004-09-01

    Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93-101 micromol g(-1) dry weight of beta-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants.

  1. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  2. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  3. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  4. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  5. Complete plastid genome sequencing of Trochodendraceae reveals a significant expansion of the inverted repeat and suggests a Paleogene divergence between the two extant species.

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    Yan-xia Sun

    Full Text Available The early-diverging eudicot order Trochodendrales contains only two monospecific genera, Tetracentron and Trochodendron. Although an extensive fossil record indicates that the clade is perhaps 100 million years old and was widespread throughout the Northern Hemisphere during the Paleogene and Neogene, the two extant genera are both narrowly distributed in eastern Asia. Recent phylogenetic analyses strongly support a clade of Trochodendrales, Buxales, and Gunneridae (core eudicots, but complete plastome analyses do not resolve the relationships among these groups with strong support. However, plastid phylogenomic analyses have not included data for Tetracentron. To better resolve basal eudicot relationships and to clarify when the two extant genera of Trochodendrales diverged, we sequenced the complete plastid genome of Tetracentron sinense using Illumina technology. The Tetracentron and Trochodendron plastomes possess the typical gene content and arrangement that characterize most angiosperm plastid genomes, but both genomes have the same unusual ∼4 kb expansion of the inverted repeat region to include five genes (rpl22, rps3, rpl16, rpl14, and rps8 that are normally found in the large single-copy region. Maximum likelihood analyses of an 83-gene, 88 taxon angiosperm data set yield an identical tree topology as previous plastid-based trees, and moderately support the sister relationship between Buxaceae and Gunneridae. Molecular dating analyses suggest that Tetracentron and Trochodendron diverged between 44-30 million years ago, which is congruent with the fossil record of Trochodendrales and with previous estimates of the divergence time of these two taxa. We also characterize 154 simple sequence repeat loci from the Tetracentron sinense and Trochodendron aralioides plastomes that will be useful in future studies of population genetic structure for these relict species, both of which are of conservation concern.

  6. Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

    Science.gov (United States)

    dePamphilis, Claude W.; Young, Nelson D.; Wolfe, Andrea D.

    1997-01-01

    The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism. PMID:9207097

  7. Complete plastid genomes from Ophioglossum californicum, Psilotum nudum, and Equisetum hyemale reveal an ancestral land plant genome structure and resolve the position of Equisetales among monilophytes

    Directory of Open Access Journals (Sweden)

    Grewe Felix

    2013-01-01

    Full Text Available Abstract Background Plastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic. Results In order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales, Ophioglossum californicum (Ophioglossales, and Psilotum nudum (Psilotales. A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida. Conclusions Although molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic

  8. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  9. The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

    International Nuclear Information System (INIS)

    Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa; Rademacher, Thomas; Lampel, Johannes; Eudes, François; Vitale, Alessandro; Stoger, Eva

    2014-01-01

    Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

  10. The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence

    Energy Technology Data Exchange (ETDEWEB)

    Hofbauer, Anna; Peters, Jenny; Arcalis, Elsa [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria); Rademacher, Thomas [Institute of Molecular Biotechnology, RWTH Aachen University, Aachen (Germany); Lampel, Johannes [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria); Eudes, François [Agriculture and Agri-Food Canada, Lethbridge, AB (Canada); Vitale, Alessandro [Institute of Agricultural Biology and Biotechnology, National Research Council (CNR), Milan (Italy); Stoger, Eva, E-mail: eva.stoger@boku.ac.at [Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (Austria)

    2014-12-11

    Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

  11. Methods for RNA Analysis

    DEFF Research Database (Denmark)

    Olivarius, Signe

    of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

  12. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  13. The Bryopsis hypnoides plastid genome: multimeric forms and complete nucleotide sequence.

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    Fang Lü

    Full Text Available BACKGROUND: Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga. PRINCIPAL FINDINGS: A total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp, arrangement, and inverted-repeat (IR-lacking structure of the B. hypnoides chloroplast DNA (cpDNA closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support. CONCLUSION: All of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.

  14. DNA barcoding based on plastid matK and RNA polymerase for assessing the genetic identity of date (Phoenix dactylifera L.) cultivars.

    Science.gov (United States)

    Enan, M R; Ahamed, A

    2014-02-14

    The cultivated date palm is the most agriculturally important species of the Arecaceae family. The standard chloroplast DNA barcode for land plants recommended by the Consortium for the Barcode of Life plant working group needs to be evaluated for a wide range of plant species. Therefore, we assessed the potential of the matK and rpoC1 markers for the authentication of date cultivars. There is not one universal method to authenticate date cultivars. In this study, 11 different date cultivars were sequenced and analyzed for matK and rpoC1 genes by using bioinformatic tools to establish a cultivar-specific molecular monogram. The chloroplast matK marker was more informative than the rpoC1 chloroplast DNA markers. Phylogenetic trees were constructed on the basis of the matK and rpoC1 sequences, and the results suggested that matK alone or in combination with rpoC1 can be used for determining the levels of genetic variation and for barcoding.

  15. Variability among the Most Rapidly Evolving Plastid Genomic Regions is Lineage-Specific: Implications of Pairwise Genome Comparisons in Pyrus (Rosaceae) and Other Angiosperms for Marker Choice

    Science.gov (United States)

    Ter-Voskanyan, Hasmik; Allgaier, Martin; Borsch, Thomas

    2014-01-01

    Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae)—a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC–trnV, trnR–atpA, ndhF–rpl32, psbM–trnD, and trnQ–rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters). Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid), Olea (asterids) and Cymbidium (monocots) showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF–rpl32 and trnK–rps16) were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations

  16. Variability among the most rapidly evolving plastid genomic regions is lineage-specific: implications of pairwise genome comparisons in Pyrus (Rosaceae and other angiosperms for marker choice.

    Directory of Open Access Journals (Sweden)

    Nadja Korotkova

    Full Text Available Plastid genomes exhibit different levels of variability in their sequences, depending on the respective kinds of genomic regions. Genes are usually more conserved while noncoding introns and spacers evolve at a faster pace. While a set of about thirty maximum variable noncoding genomic regions has been suggested to provide universally promising phylogenetic markers throughout angiosperms, applications often require several regions to be sequenced for many individuals. Our project aims to illuminate evolutionary relationships and species-limits in the genus Pyrus (Rosaceae-a typical case with very low genetic distances between taxa. In this study, we have sequenced the plastid genome of Pyrus spinosa and aligned it to the already available P. pyrifolia sequence. The overall p-distance of the two Pyrus genomes was 0.00145. The intergenic spacers between ndhC-trnV, trnR-atpA, ndhF-rpl32, psbM-trnD, and trnQ-rps16 were the most variable regions, also comprising the highest total numbers of substitutions, indels and inversions (potentially informative characters. Our comparative analysis of further plastid genome pairs with similar low p-distances from Oenothera (representing another rosid, Olea (asterids and Cymbidium (monocots showed in each case a different ranking of genomic regions in terms of variability and potentially informative characters. Only two intergenic spacers (ndhF-rpl32 and trnK-rps16 were consistently found among the 30 top-ranked regions. We have mapped the occurrence of substitutions and microstructural mutations in the four genome pairs. High AT content in specific sequence elements seems to foster frequent mutations. We conclude that the variability among the fastest evolving plastid genomic regions is lineage-specific and thus cannot be precisely predicted across angiosperms. The often lineage-specific occurrence of stem-loop elements in the sequences of introns and spacers also governs lineage-specific mutations. Sequencing

  17. PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Anat Florentin

    2017-11-01

    Full Text Available Summary: The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex. : Plasmodium falciparum contains a unique organelle, the apicoplast. Using genetic and phenotypic assays, Florentin et al. characterize the apicoplast Clp chaperone and protease. They find that the chaperone is essential for protease stability and that together they function to maintain organelle integrity and segregation into daughter cells. Keywords: malaria, Plasmodium, apicoplast, IPP, Clp, chaperone, caseinolytic protease

  18. The Plant Immunity Regulating F-Box Protein CPR1 Supports Plastid Function in Absence of Pathogens

    Directory of Open Access Journals (Sweden)

    Christiane Hedtmann

    2017-09-01

    Full Text Available The redox imbalanced 6 mutant (rimb6 of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR GENES 1, CPR1 (At4g12560, leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.

  19. A Set of Plastid Loci for Use in Multiplex Fragment Length Genotyping for Intraspecific Variation in Pinus (Pinaceae

    Directory of Open Access Journals (Sweden)

    Austin M. Wofford

    2014-04-01

    Full Text Available Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences ofycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. Results: The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. Discussion: The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group.

  20. A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae)1

    Science.gov (United States)

    Wofford, Austin M.; Finch, Kristen; Bigott, Adam; Willyard, Ann

    2014-01-01

    • Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. • Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences of ycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. • Results: The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. • Discussion: The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group. PMID:25202625

  1. Phylogeny of Eleusine (Poaceae: Chloridoideae) based on nuclear ITS and plastid trnT-trnF sequences.

    Science.gov (United States)

    Neves, Susana S; Swire-Clark, Ginger; Hilu, Khidir W; Baird, Wm Vance

    2005-05-01

    Phylogenetic relationships in the genus Eleusine (Poaceae: Chloridoideae) were investigated using nuclear ITS and plastid trnT-trnF sequences. Separate and combined data sets were analyzed using parsimony, distance, and likelihood based methods, including Bayesian. Data congruence was examined using character and topological measures. Significant data heterogeneity was detected, but there was little conflict in the topological substructure measures for triplets and quartets, and resolution and clade support increased in the combined analysis. Data incongruence may be a result of noise and insufficient information in the slower evolving trnT-trnF. Monophyly of Eleusine is strongly supported in all analyses, but basal relationships in the genus remain uncertain. There is good support for a CAIK clade (E. coracana subsp. coracana and africana, E. indica, and E. kigeziensis), with E. tristachya as its sister group. Two putative ITS homeologues (A and B loci) were identified in the allotetraploid E. coracana; the 'B' locus sequence type was not found in the remaining species. Eleusine coracana and its putative 'A' genome donor, the diploid E. indica, are confirmed close allies, but sequence data contradicts the hypothesis that E. floccifolia is its second genome donor. The 'B' genome donor remains unidentified and may be extinct.

  2. The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

    Science.gov (United States)

    Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

    2017-04-28

    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Nuclear and plastid markers reveal the persistence of genetic identity: a new perspective on the evolutionary history of Petunia exserta.

    Science.gov (United States)

    Segatto, Ana Lúcia Anversa; Cazé, Ana Luíza Ramos; Turchetto, Caroline; Klahre, Ulrich; Kuhlemeier, Cris; Bonatto, Sandro Luis; Freitas, Loreta Brandão

    2014-01-01

    Recently divergent species that can hybridize are ideal models for investigating the genetic exchanges that can occur while preserving the species boundaries. Petunia exserta is an endemic species from a very limited and specific area that grows exclusively in rocky shelters. These shaded spots are an inhospitable habitat for all other Petunia species, including the closely related and widely distributed species P. axillaris. Individuals with intermediate morphologic characteristics have been found near the rocky shelters and were believed to be putative hybrids between P. exserta and P. axillaris, suggesting a situation where Petunia exserta is losing its genetic identity. In the current study, we analyzed the plastid intergenic spacers trnS/trnG and trnH/psbA and six nuclear CAPS markers in a large sampling design of both species to understand the evolutionary process occurring in this biological system. Bayesian clustering methods, cpDNA haplotype networks, genetic diversity statistics, and coalescence-based analyses support a scenario where hybridization occurs while two genetic clusters corresponding to two species are maintained. Our results reinforce the importance of coupling differentially inherited markers with an extensive geographic sample to assess the evolutionary dynamics of recently diverged species that can hybridize. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

  5. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  6. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  7. Plastid phylogenomics of the cool-season grass subfamily: clarification of relationships among early-diverging tribes.

    Science.gov (United States)

    Saarela, Jeffery M; Wysocki, William P; Barrett, Craig F; Soreng, Robert J; Davis, Jerrold I; Clark, Lynn G; Kelchner, Scot A; Pires, J Chris; Edger, Patrick P; Mayfield, Dustin R; Duvall, Melvin R

    2015-05-04

    Whole plastid genomes are being sequenced rapidly from across the green plant tree of life, and phylogenetic analyses of these are increasing resolution and support for relationships that have varied among or been unresolved in earlier single- and multi-gene studies. Pooideae, the cool-season grass lineage, is the largest of the 12 grass subfamilies and includes important temperate cereals, turf grasses and forage species. Although numerous studies of the phylogeny of the subfamily have been undertaken, relationships among some 'early-diverging' tribes conflict among studies, and some relationships among subtribes of Poeae have not yet been resolved. To address these issues, we newly sequenced 25 whole plastomes, which showed rearrangements typical of Poaceae. These plastomes represent 9 tribes and 11 subtribes of Pooideae, and were analysed with 20 existing plastomes for the subfamily. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) robustly resolve most deep relationships in the subfamily. Complete plastome data provide increased nodal support compared with protein-coding data alone at nodes that are not maximally supported. Following the divergence of Brachyelytrum, Phaenospermateae, Brylkinieae-Meliceae and Ampelodesmeae-Stipeae are the successive sister groups of the rest of the subfamily. Ampelodesmeae are nested within Stipeae in the plastome trees, consistent with its hybrid origin between a phaenospermatoid and a stipoid grass (the maternal parent). The core Pooideae are strongly supported and include Brachypodieae, a Bromeae-Triticeae clade and Poeae. Within Poeae, a novel sister group relationship between Phalaridinae and Torreyochloinae is found, and the relative branching order of this clade and Aveninae, with respect to an Agrostidinae-Brizinae clade, are discordant between MP and ML/BI trees. Maximum likelihood and Bayesian analyses strongly support Airinae and Holcinae as the successive sister groups of a Dactylidinae

  8. A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Justin D. Fellows

    2017-06-01

    Full Text Available Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL, an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

  9. Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO2 fixation in wheat (Triticum aestivum) following heat stress.

    Science.gov (United States)

    Fu, Jianming; Momcilović, Ivana; Clemente, Thomas E; Nersesian, Natalya; Trick, Harold N; Ristic, Zoran

    2008-10-01

    Heat stress is a major constraint to wheat production and negatively impacts grain quality, causing tremendous economic losses, and may become a more troublesome factor due to global warming. At the cellular level, heat stress causes denaturation and aggregation of proteins and injury to membranes leading to alterations in metabolic fluxes. Protein aggregation is irreversible, and protection of proteins from thermal aggregation is a strategy a cell uses to tolerate heat stress. Here we report on the development of transgenic wheat (Triticum aestivum) events, expressing a maize gene coding for plastidal protein synthesis elongation factor (EF-Tu), which, compared to non-transgenic plants, display reduced thermal aggregation of leaf proteins, reduced heat injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation after exposure to heat stress. The results support the concept that EF-Tu ameliorates negative effects of heat stress by acting as a molecular chaperone. This is the first demonstration of the introduction of a plastidal EF-Tu in plants that leads to protection against heat injury and enhanced photosynthesis after heat stress. This is also the first demonstration that a gene other than HSP gene can be used for improvement of heat tolerance and that the improvement is possible in a species that has a complex genome, hexaploid wheat. The results strongly suggest that heat tolerance of wheat, and possibly other crop plants, can be improved by modulating expression of plastidal EF-Tu and/or by selection of genotypes with increased endogenous levels of this protein.

  10. A multi-locus plastid phylogenetic analysis of the pantropical genus Diospyros (Ebenaceae), with an emphasis on the radiation and biogeographic origins of the New Caledonian endemic species

    OpenAIRE

    Duangjai, S.; Samuel, R.; Munzinger, Jérôme; Forest, F.; Wallnofer, B.; Barfuss, M.H.J.; Fischer, G.; Chase, M. W.

    2009-01-01

    We aimed to clarify phylogenetic relationships within the pantropical genus Diospyros (Ebenaceae sensu lato), and ascertain biogeographical patterns in the New Caledonian endemic species. We used DNA sequences from eight plastid regions (rbcL, atpB, matK, ndhF, trnK intron, trnL intron, trnL-trnF spacer, and trnS-trnG spacer) and included 149 accessions representing 119 Diospyros species in our analysis. Results from this study confirmed the monophyly of Diospyros with good support and provid...

  11. The plastid genome of some eustigmatophyte algae harbours a bacteria-derived six-gene cluster for biosynthesis of a novel secondary metabolite

    Czech Academy of Sciences Publication Activity Database

    Yurchenko, T.; Ševčíková, T.; Strnad, Hynek; Butenko, A.; Eliáš, M.

    2016-01-01

    Roč. 6, č. 11 (2016), č. článku 160249. ISSN 2046-2441 R&D Projects: GA ČR GA13-33039S; GA MŠk(CZ) ED2.1.00/19.0388; GA MŠk(CZ) LO1208 EU Projects: European Commission 642575 Institutional support: RVO:68378050 Keywords : Eustigmatophyceae * horizontal gene transfer * plastid genome * secondary metabolism * sugar phosphate cyclase superfamily * UbiA superfamily Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.481, year: 2016

  12. FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division

    Directory of Open Access Journals (Sweden)

    Shin-Ya eMiyagishima

    2014-09-01

    Full Text Available The chloroplast division machinery is a mixture of a stromal FtsZ-based complex descended from a cyanobacterial ancestor of chloroplasts and a cytosolic dynamin-related protein (DRP 5B-based complex derived from the eukaryotic host. Molecular genetic studies have shown that each component of the division machinery is normally essential for normal chloroplast division. However, several exceptions have been found. In the absence of the FtsZ ring, nonphotosynthetic plastids are able to proliferate, likely by elongation and budding. Depletion of DRP5B impairs, but does not stop chloroplast division. Chloroplasts in glaucophytes, which possesses a peptidoglycan (PG layer, divide without DRP5B. Certain parasitic eukaryotes possess nonphotosynthetic plastids of secondary endosymbiotic origin, but neither FtsZ nor DRP5B is encoded in their genomes. Elucidation of the FtsZ- and/or DRP5B-less chloroplast division mechanism will lead to a better understanding of the function and evolution of the chloroplast division machinery and the finding of the as-yet-unknown mechanism that is likely involved in chloroplast division. Recent studies have shown that FtsZ was lost from a variety of prokaryotes, many of which lost PG by regressive evolution. In addition, even some of the FtsZ-bearing bacteria are able to divide when FtsZ and PG are depleted experimentally. In some cases, alternative mechanisms for cell division, such as budding by an increase of the cell surface-to-volume ratio, are proposed. Although PG is believed to have been lost from chloroplasts other than in glaucophytes, there is some indirect evidence for the existence of PG in chloroplasts. Such information is also useful for understanding how nonphotosynthetic plastids are able to divide in FtsZ-depleted cells and the reason for the retention of FtsZ in chloroplast division. Here we summarize information to facilitate analyses of FtsZ- and/or DRP5B-less chloroplast and nonphotosynthetic plastid

  13. Assembling RNA Nanoparticles.

    Science.gov (United States)

    Xiao, Shou-Jun

    2017-01-01

    RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

  14. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  15. Shapes of interacting RNA complexes

    DEFF Research Database (Denmark)

    Fu, Benjamin Mingming; Reidys, Christian

    2014-01-01

    Shapes of interacting RNA complexes are studied using a filtration via their topological genus. A shape of an RNA complex is obtained by (iteratively) collapsing stacks and eliminating hairpin loops.This shape-projection preserves the topological core of the RNA complex and for fixed topological...... genus there are only finitely many such shapes. Our main result is a new bijection that relates the shapes of RNA complexes with shapes of RNA structures. This allows to compute the shape polynomial of RNA complexes via the shape polynomial of RNA structures. We furthermore present a linear time uniform...... sampling algorithm for shapes of RNA complexes of fixed topological genus....

  16. Remote Network Access (RNA)

    National Research Council Canada - National Science Library

    2002-01-01

    .... Remote Network Access (RNA) includes or is associated with all communication devices/software, firewalls, intrusion detection systems and virus protection applications to ensure security of the OIG, DoD, Network from remote...

  17. RNA/PNA Approach

    Indian Academy of Sciences (India)

    In this approach we want to develop structural analogue of the leader that might have higher affinity towards the Phosphoprotein, but would impair the dimerization process and viral leader RNA binding.

  18. Contradiction between plastid gene transcription and function due to complex posttranscriptional splicing: an exemplary study of ycf15 function and evolution in angiosperms.

    Directory of Open Access Journals (Sweden)

    Chao Shi

    Full Text Available Plant chloroplast genes are usually co-transcribed while its posttranscriptional splicing is fairly complex and remains largely unsolved. On basis of sequencing the three complete Camellia (Theaceae chloroplast genomes for the first time, we comprehensively analyzed the evolutionary patterns of ycf15, a plastid gene quite paradoxical in terms of its function and evolution, along the inferred angiosperm phylogeny. Although many species in separate lineages including the three species reported here contained an intact ycf15 gene in their chloroplast genomes, the phylogenetic mixture of both intact and obviously disabled ycf15 genes imply that they are all non-functional. Both intracellular gene transfer (IGT and horizontal gene transfer (HGT failed to explain such distributional anomalies. While, transcriptome analyses revealed that ycf15 was transcribed as precursor polycistronic transcript which contained ycf2, ycf15 and antisense trnL-CAA. The transcriptome assembly was surprisingly found to cover near the complete Camellia chloroplast genome. Many non-coding regions including pseudogenes were mapped by multiple transcripts, indicating the generality of pseudogene transcriptions. Our results suggest that plastid DNA posttranscriptional splicing may involve complex cleavage of non-functional genes.

  19. Cell-specific CO2 fixation rates of two distinct groups of plastidic protists in the Atlantic Ocean remain unchanged after nutrient addition.

    Science.gov (United States)

    Grob, Carolina; Jardillier, Ludwig; Hartmann, Manuela; Ostrowski, Martin; Zubkov, Mikhail V; Scanlan, David J

    2015-04-01

    To assess the role of open-ocean ecosystems in global CO2 fixation, we investigated how picophytoplankton, which dominate primary production, responded to episodic increases in nutrient availability. Previous experiments have shown nitrogen alone, or in combination with phosphorus or iron, to be the proximate limiting nutrient(s) for total phytoplankton grown over several days. Much less is known about how nutrient upshift affects picophytoplankton CO2 fixation over the duration of the light period. To address this issue, we performed a series of small volume (8-60 ml) - short term (10-11 h) nutrient addition experiments in different regions of the Atlantic Ocean using NH4 Cl, FeCl3 , K medium, dust and nutrient-rich water from 300 m depth. We found no significant nutrient stimulation of group-specific CO2 fixation rates of two taxonomically and size-distinct groups of plastidic protists. The above was true regardless of the region sampled or nutrient added, suggesting that this is a generic phenomenon. Our findings show that at least in the short term (i.e. daylight period), nutrient availability does not limit CO2 fixation by the smallest plastidic protists, while their taxonomic composition does not determine their response to nutrient addition. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  20. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

    2009-01-01

    to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  1. A ribosome without RNA

    Directory of Open Access Journals (Sweden)

    Harold S Bernhardt

    2015-11-01

    Full Text Available It was Francis Crick who first asked why the ribosome contains so much RNA, and discussed the implications of this for the direct flow of genetic information from DNA to protein. Remarkable advances in our understanding of the ribosome and protein synthesis, including the recent publication of two mammalian mitochondrial ribosome structures, have shed new light on this intriguing aspect of evolution in molecular biology. We examine here whether RNA is indispensable for coded protein synthesis, or whether an all-protein ‘ribosome’ (or ‘synthosome’ might be possible, with a protein enzyme catalyzing peptide synthesis, and release factor-like protein adaptors able to read a message composed of deoxyribonucleotides. We also compare the RNA world hypothesis with the alternative ‘proteins first’ hypothesis in terms of their different understandings of the evolution of the ribosome, and whether this might have been preceded by an ancestral form of nonribosomal peptide synthesis catalyzed by protein enzymes.

  2. Pyrite footprinting of RNA

    International Nuclear Information System (INIS)

    Schlatterer, Jörg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

    2012-01-01

    Highlights: ► RNA structure is mapped by pyrite mediated · OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High · OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ( · OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS 2 ) can produce sufficient · OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to · OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated · OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

  3. RNA Regulation of Estrogen

    Science.gov (United States)

    2010-08-01

    Berglund, Rodger Voelker, Paul Barber and Julien Diegel 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING...estrogen  receptors  [reviewed  in  (3,  4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA...Mog myelin oligodendrocyte glycoprotein 6.06 207115_x_at mbtd1 mbt domain containing 1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at

  4. RNA Regulation by Estrogen

    Science.gov (United States)

    2011-08-01

    Julien Diegel, Amy Mahady, and Micah Bodner 5e. TASK NUMBER E-Mail: aberglund@molbio.uoregon.edu 5f. WORK UNIT NUMBER 7. PERFORMING...4)],  also   functions   by  interacting  directly  with  RNA  to  alter  RNA  processing  events  such  as  splicing...1 6.06 208004_at Prol1 proline rich, lacrimal 1 6.06 205247_at NOTCH4 Notch homolog 4 (Drosophila) 6.06 211203_s_at Cntn1 contactin 1 6.06 220689_at

  5. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.......Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

  6. RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

    Directory of Open Access Journals (Sweden)

    Andronescu Mirela

    2008-08-01

    Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

  7. An Arabidopsis introgression zone studied at high spatio-temporal resolution: interglacial and multiple genetic contact exemplified using whole nuclear and plastid genomes.

    Science.gov (United States)

    Hohmann, Nora; Koch, Marcus A

    2017-10-23

    Gene flow between species, across ploidal levels, and even between evolutionary lineages is a common phenomenon in the genus Arabidopsis. However, apart from two genetically fully stabilized allotetraploid species that have been investigated in detail, the extent and temporal dynamics of hybridization are not well understood. An introgression zone, with tetraploid A. arenosa introgressing into A. lyrata subsp. petraea in the Eastern Austrian Forealps and subsequent expansion towards pannonical lowlands, was described previously based on morphological observations as well as molecular data using microsatellite and plastid DNA markers. Here we investigate the spatio-temporal context of this suture zone, making use of the potential of next-generation sequencing and whole-genome data. By utilizing a combination of nuclear and plastid genomic data, the extent, direction and temporal dynamics of gene flow are elucidated in detail and Late Pleistocene evolutionary processes are resolved. Analysis of nuclear genomic data significantly recognizes the clinal structure of the introgression zone, but also reveals that hybridization and introgression is more common and substantial than previously thought. Also tetraploid A. lyrata and A. arenosa subsp. borbasii from outside the previously defined suture zone show genomic signals of past introgression. A. lyrata is shown to serve usually as the maternal parent in these hybridizations, but one exception is identified from plastome-based phylogenetic reconstruction. Using plastid phylogenomics with secondary time calibration, the origin of A. lyrata and A. arenosa lineages is pre-dating the last three glaciation complexes (approx. 550,000 years ago). Hybridization and introgression followed during the last two glacial-interglacial periods (since approx. 300,000 years ago) with later secondary contact at the northern and southern border of the introgression zone during the Holocene. Footprints of adaptive introgression in the

  8. Studying RNA-protein interactions in vivo by RNA immunoprecipitation

    DEFF Research Database (Denmark)

    Selth, Luke A; Close, Pierre; Svejstrup, Jesper Q

    2011-01-01

    and have significant effects on gene expression. RNA immunoprecipitation (RIP) is a powerful technique used to detect direct and indirect interactions between individual proteins and specific RNA molecules in vivo. Here, we describe RIP methods for both yeast and mammalian cells.......The crucial roles played by RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of transcription, have become increasingly evident. Moreover, other factors that do not directly interact with RNA molecules can nevertheless function proximally to RNA polymerases...

  9. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  10. The RNA gene information: retroelement-microRNA entangling as the RNA quantum code.

    Science.gov (United States)

    Fujii, Yoichi Robertus

    2013-01-01

    MicroRNA (miRNA) and retroelements may be a master of regulator in our life, which are evolutionally involved in the origin of species. To support the Darwinism from the aspect of molecular evolution process, it has tremendously been interested in the molecular information of naive RNA. The RNA wave model 2000 consists of four concepts that have altered from original idea of the miRNA genes for crosstalk among embryonic stem cells, their niche cells, and retroelements as a carrier vesicle of the RNA genes. (1) the miRNA gene as a mobile genetic element induces transcriptional and posttranscriptional silencing via networking-processes (no hierarchical architecture); (2) the RNA information supplied by the miRNA genes expands to intracellular, intercellular, intraorgan, interorgan, intraspecies, and interspecies under the cycle of life into the global environment; (3) the mobile miRNAs can self-proliferate; and (4) cells contain two types information as resident and genomic miRNAs. Based on RNA wave, we have developed an interest in investigation of the transformation from RNA information to quantum bits as physicochemical characters of RNA with the measurement of RNA electron spin. When it would have been given that the fundamental bases for the acquired characters in genetics can be controlled by RNA gene information, it may be available to apply for challenging against RNA gene diseases, such as stress-induced diseases.

  11. Plant RNA Regulatory Network and RNA Granules in Virus Infection

    Directory of Open Access Journals (Sweden)

    Kristiina Mäkinen

    2017-12-01

    Full Text Available Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and

  12. Plant RNA Regulatory Network and RNA Granules in Virus Infection.

    Science.gov (United States)

    Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

    2017-01-01

    Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual

  13. RNA-Catalyzed Polymerization and Replication of RNA

    Science.gov (United States)

    Horning, D. P.; Samantha, B.; Tjhung, K. F.; Joyce, G. F.

    2017-07-01

    In an effort to reconstruct RNA-based life, in vitro evolution was used to obtain an RNA polymerase ribozyme that can synthesize a variety of complex functional RNAs and can catalyze the exponential amplification of short RNAs.

  14. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

    Directory of Open Access Journals (Sweden)

    Amarjeet Kumar Singh

    Full Text Available Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp, which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

  15. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink.

    Science.gov (United States)

    Stepien, Piotr; Johnson, Giles N

    2009-02-01

    The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.

  16. Natural RNA circles function as efficient microRNA sponges

    DEFF Research Database (Denmark)

    Hansen, Thomas Birkballe; Jensen, Trine I; Clausen, Bettina Hjelm

    2013-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called comp......MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so......-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more...... sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA....

  17. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double

  18. RNA Interference - Towards RNA becoming a Medicine -42 ...

    Indian Academy of Sciences (India)

    research. A brief history of the development ofRNAi is shown in. Box 2. Mechanism of ... new RNA strand using target RNA as the template and thereby converting it ... thought to excise precursor stRNA from their -70 nt stem loop precursor to ...

  19. Semiautomated improvement of RNA alignments

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

    2007-01-01

    connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster......: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture...

  20. Comparative RNA genomics

    DEFF Research Database (Denmark)

    Backofen, Rolf; Gorodkin, Jan; Hofacker, Ivo L.

    2018-01-01

    Over the last two decades it has become clear that RNA is much more than just a boring intermediate in protein expression. Ancient RNAs still appear in the core information metabolism and comprise a surprisingly large component in bacterial gene regulation. A common theme with these types of mostly...... small RNAs is their reliance of conserved secondary structures. Large scale sequencing projects, on the other hand, have profoundly changed our understanding of eukaryotic genomes. Pervasively transcribed, they give rise to a plethora of large and evolutionarily extremely flexible noncoding RNAs...... that exert a vastly diverse array of molecule functions. In this chapter we provide a—necessarily incomplete—overview of the current state of comparative analysis of noncoding RNAs, emphasizing computational approaches as a means to gain a global picture of the modern RNA world....

  1. MicroRNA from tuberculosis RNA: A bioinformatics study

    OpenAIRE

    Wiwanitkit, Somsri; Wiwanitkit, Viroj

    2012-01-01

    The role of microRNA in the pathogenesis of pulmonary tuberculosis is the interesting topic in chest medicine at present. Recently, it was proposed that the microRNA can be a useful biomarker for monitoring of pulmonary tuberculosis and might be the important part in pathogenesis of disease. Here, the authors perform a bioinformatics study to assess the microRNA within known tuberculosis RNA. The microRNA part can be detected and this can be important key information in further study of the p...

  2. RNA binding and replication by the poliovirus RNA polymerase

    International Nuclear Information System (INIS)

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

  3. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

    International Nuclear Information System (INIS)

    Bodkin, D.K.

    1985-01-01

    RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

  4. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    Science.gov (United States)

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

  5. RNA meets disease in paradise.

    Science.gov (United States)

    Winter, Julia; Roth, Anna; Diederichs, Sven

    2011-01-01

    Getting off the train in Jena-Paradies, 60 participants joined for the 12 (th) Young Scientist Meeting of the German Society for Cell Biology (DGZ) entitled "RNA & Disease". Excellent speakers from around the world, graduate students, postdocs and young group leaders enjoyed a meeting in a familiar atmosphere to exchange inspiring new data and vibrant scientific discussions about the fascinating history and exciting future of non-coding RNA research including microRNA, piRNA and long non-coding RNA as well as their function in cancer, diabetes and neurodegenerative diseases.

  6. From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

    Science.gov (United States)

    Michelini, Flavia; Jalihal, Ameya P; Francia, Sofia; Meers, Chance; Neeb, Zachary T; Rossiello, Francesca; Gioia, Ubaldo; Aguado, Julio; Jones-Weinert, Corey; Luke, Brian; Biamonti, Giuseppe; Nowacki, Mariusz; Storici, Francesca; Carninci, Piero; Walter, Nils G; Fagagna, Fabrizio d'Adda di

    2018-03-30

    Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

  7. Transfer RNA and human disease

    Directory of Open Access Journals (Sweden)

    Jamie A Abbott

    2014-06-01

    Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  8. Transfer RNA and human disease.

    Science.gov (United States)

    Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  9. RNA Thermodynamic Structural Entropy.

    Science.gov (United States)

    Garcia-Martin, Juan Antonio; Clote, Peter

    2015-01-01

    Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  10. RNA Thermodynamic Structural Entropy.

    Directory of Open Access Journals (Sweden)

    Juan Antonio Garcia-Martin

    Full Text Available Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs. However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  11. Identifying microRNA/mRNA dysregulations in ovarian cancer.

    Science.gov (United States)

    Miles, Gregory D; Seiler, Michael; Rodriguez, Lorna; Rajagopal, Gunaretnam; Bhanot, Gyan

    2012-03-27

    MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Our findings identify

  12. Intricate patterns of phylogenetic relationships in the olive family as inferred from multi-locus plastid and nuclear DNA sequence analyses: a close-up on Chionanthus and Noronhia (Oleaceae).

    Science.gov (United States)

    Hong-Wa, Cynthia; Besnard, Guillaume

    2013-05-01

    Noronhia represents the most successful radiation of the olive family (Oleaceae) in Madagascar with more than 40 named endemic species distributed in all ecoregions from sea level to high mountains. Its position within the subtribe Oleinae has, however, been largely unresolved and its evolutionary history has remained unexplored. In this study, we generated a dataset of plastid (trnL-F, trnT-L, trnS-G, trnK-matK) and nuclear (internal transcribed spacer [ITS]) DNA sequences to infer phylogenetic relationships within Oleinae and to examine evolutionary patterns within Noronhia. Our sample included most species of Noronhia and representatives of the ten other extant genera within the subtribe with an emphasis on Chionanthus. Bayesian inferences and maximum likelihood analyses of plastid and nuclear data indicated several instances of paraphyly and polyphyly within Oleinae, with some geographic signal. Both plastid and ITS data showed a polyphyletic Noronhia that included Indian Ocean species of Chionanthus. They also found close relationships between Noronhia and African Chionanthus. However, the plastid data showed little clear differentiation between Noronhia and the African Chionanthus whereas relationships suggested by the nuclear ITS data were more consistent with taxonomy and geography. We used molecular dating to discriminate between hybridization and lineage sorting/gene duplication as alternative explanations for these topological discordances and to infer the biogeographic history of Noronhia. Hybridization between African Chionanthus and Noronhia could not be ruled out. However, Noronhia has long been established in Madagascar after a likely Cenozoic dispersal from Africa, suggesting any hybridization between representatives of African and Malagasy taxa was ancient. In any case, the African and Indian Ocean Chionanthus and Noronhia together formed a strongly supported monophyletic clade distinct and distant from other Chionanthus, which calls for a revised

  13. RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

    International Nuclear Information System (INIS)

    Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E.; Eghbalnia, Hamid R.

    2012-01-01

    The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ( 1 H– 15 N 2D HMQC) and proton–proton nuclear Overhauser enhancement spectroscopy ( 1 H– 1 H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a

  14. RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

    Energy Technology Data Exchange (ETDEWEB)

    Bahrami, Arash; Clos, Lawrence J.; Markley, John L.; Butcher, Samuel E. [National Magnetic Resonance Facility at Madison (United States); Eghbalnia, Hamid R., E-mail: eghbalhd@uc.edu [University of Cincinnati, Department of Molecular and Cellular Physiology (United States)

    2012-04-15

    The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ({sup 1}H-{sup 15}N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ({sup 1}H-{sup 1}H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino

  15. RNA SURVEILLANCE– AN EMERGING ROLE FOR RNA REGULATORY NETWORKS IN AGING

    OpenAIRE

    Montano, Monty; Long, Kimberly

    2010-01-01

    In this review, we describe recent advances in the field of RNA regulatory biology and relate these advances to aging science. We introduce a new term, RNA surveillance, an RNA regulatory process that is conserved in metazoans, and describe how RNA surveillance represents molecular cross-talk between two emerging RNA regulatory systems – RNA interference and RNA editing. We discuss how RNA surveillance mechanisms influence mRNA and microRNA expression and activity during lifespan. Additionall...

  16. On RNA-RNA interaction structures of fixed topological genus.

    Science.gov (United States)

    Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

    2015-04-01

    Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. antaRNA: ant colony-based RNA sequence design.

    Science.gov (United States)

    Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

    2015-10-01

    RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  18. Multiple Events of Allopolyploidy in the Evolution of the Racemose Lineages in Prunus (Rosaceae Based on Integrated Evidence from Nuclear and Plastid Data.

    Directory of Open Access Journals (Sweden)

    Liang Zhao

    Full Text Available Prunus is an economically important genus well-known for cherries, plums, almonds, and peaches. The genus can be divided into three major groups based on inflorescence structure and ploidy levels: (1 the diploid solitary-flower group (subg. Prunus, Amygdalus and Emplectocladus; (2 the diploid corymbose group (subg. Cerasus; and (3 the polyploid racemose group (subg. Padus, subg. Laurocerasus, and the Maddenia group. The plastid phylogeny suggests three major clades within Prunus: Prunus-Amygdalus-Emplectocladus, Cerasus, and Laurocerasus-Padus-Maddenia, while nuclear ITS trees resolve Laurocerasus-Padus-Maddenia as a paraphyletic group. In this study, we employed sequences of the nuclear loci At103, ITS and s6pdh to explore the origins and evolution of the racemose group. Two copies of the At103 gene were identified in Prunus. One copy is found in Prunus species with solitary and corymbose inflorescences as well as those with racemose inflorescences, while the second copy (II is present only in taxa with racemose inflorescences. The copy I sequences suggest that all racemose species form a paraphyletic group composed of four clades, each of which is definable by morphology and geography. The tree from the combined At103 and ITS sequences and the tree based on the single gene s6pdh had similar general topologies to the tree based on the copy I sequences of At103, with the combined At103-ITS tree showing stronger support in most clades. The nuclear At103, ITS and s6pdh data in conjunction with the plastid data are consistent with the hypothesis that multiple independent allopolyploidy events contributed to the origins of the racemose group. A widespread species or lineage may have served as the maternal parent for multiple hybridizations involving several paternal lineages. This hypothesis of the complex evolutionary history of the racemose group in Prunus reflects a major step forward in our understanding of diversification of the genus and has

  19. RNA İNTERFERANS (RNAİ)

    OpenAIRE

    GÜNDOĞDU, Ramazan; ÇELİK, Venhar

    2009-01-01

    RNA interferans, uygun çift zincirli RNA’nın hücreye girdiği zaman, endojenik komplementer mRNA dizisinin parçalanmasına yol açan, transkripsiyon sonrası gen susturma mekanizmasıdır. RNA interferans, Dicer adı verilen bir RNase III enzimi tarafından çift zincirli RNA’nın küçük engelleyici RNA’lara (siRNA) kesilmesi ile başlamaktadır. Bu siRNA’lar daha sonra, bir multiprotein-RNA nükleaz kompleksi olan, RNA- indükleyici baskılama kompleksine (RISC) bağlanır. RISC, siRNA’ları komplementer mRNA’...

  20. Radiation sensitivity of messenger RNA

    International Nuclear Information System (INIS)

    Ponta, H.; Pfennig-Yeh, M.L.; Herrlich, P.; Karlsruhe Univ.; Wagner, E.F.; Schweiger, M.

    1979-01-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. (orig.) [de

  1. Radiation sensitivity of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ponta, H; Pfennig-Yeh, M L; Herrlich, P [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen; Karlsruhe Univ. (TH) (Germany, F.R.). Inst. fuer Genetik); Wagner, E F; Schweiger, M [Innsbruck Univ. (Austria). Inst. fuer Biochemie

    1979-08-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

  2. RNase-assisted RNA chromatography

    Science.gov (United States)

    Michlewski, Gracjan; Cáceres, Javier F.

    2010-01-01

    RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript. PMID:20571124

  3. RNA interference in Lepidoptera

    DEFF Research Database (Denmark)

    Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

    2011-01-01

    in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our...... experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...

  4. A multi-locus plastid phylogenetic analysis of the pantropical genus Diospyros (Ebenaceae), with an emphasis on the radiation and biogeographic origins of the New Caledonian endemic species.

    Science.gov (United States)

    Duangjai, Sutee; Samuel, Rosabelle; Munzinger, Jérôme; Forest, Félix; Wallnöfer, Bruno; Barfuss, Michael H J; Fischer, Gunter; Chase, Mark W

    2009-09-01

    We aimed to clarify phylogenetic relationships within the pantropical genus Diospyros (Ebenaceae sensulato), and ascertain biogeographical patterns in the New Caledonian endemic species. We used DNA sequences from eight plastid regions (rbcL, atpB, matK, ndhF, trnK intron, trnL intron, trnL-trnF spacer, and trnS-trnG spacer) and included 149 accessions representing 119 Diospyros species in our analysis. Results from this study confirmed the monophyly of Diospyros with good support and provided a clearer picture of the relationships within the genus than in previous studies. Evidence from phylogenetic analyses suggests that Diospyros colonized New Caledonia multiple times. The four lineages of Diospyros in New Caledonia also differ in their degree of diversification. The molecular data indicate that one lineage is paleoendemic and derived from an ancient Australian species. The other three lineages are more closely related to several Southeast Asian species; two of them are neoendemics, and one has radiated rapidly and recently.

  5. Ulva and Enteromorpha (Ulvaceae, Chlorophyta) from two sides of the Yellow Sea: analysis of nuclear rDNA ITS and plastid rbcL sequence data

    Science.gov (United States)

    Wang, Jinfeng; Li, Nan; Jiang, Peng; Boo, Sung Min; Lee, Wook Jae; Cui, Yulin; Lin, Hanzhi; Zhao, Jin; Liu, Zhengyi; Qin, Song

    2010-07-01

    Ulvacean green seaweeds are common worldwide; they formed massive green tides in the Yellow Sea in recent years, which caused marine ecological problems as well as a social issue. We investigated two major genera of the Ulvaceae, Ulva and Enteromorpha, and collected the plastid rbcL and nuclear ITS sequences of specimens of the genera in two sides of the Yellow Sea and analyzed them. Phylogenetic trees of rbcL data show the occurrence of five species of Enteromorpha ( E. compressa, E. flexuosa, E. intestinalis, E. linza and E. prolifera) and three species of Ulva ( U. pertusa, U. rigida and U. ohnoi). However, we found U. ohnoi, which is known as a subtropical to tropical species, at two sites on Jeju Island, Korea. Four ribotypes in partial sequences of 5.8S rDNA and ITS2 from E. compressa were also found. Ribotype network analysis revealed that the common ribotype, occurring in China, Korea and Europe, is connected with ribotypes from Europe and China/Japan. Although samples of the same species were collected from both sides of the Yellow Sea, intraspecific genetic polymorphism of each species was low among samples collected worldwide.

  6. Phylogeny of the Celastreae (Celastraceae) and the relationships of Catha edulis (qat) inferred from morphological characters and nuclear and plastid genes.

    Science.gov (United States)

    Simmons, Mark P; Cappa, Jennifer J; Archer, Robert H; Ford, Andrew J; Eichstedt, Dedra; Clevinger, Curtis C

    2008-08-01

    The phylogeny of Celastraceae tribe Celastreae, which includes about 350 species of trees and shrubs in 15 genera, was inferred in a simultaneous analysis of morphological characters together with nuclear (ITS and 26S rDNA) and plastid (matK, trnL-F) genes. A strong correlation was found between the geography of the species sampled and their inferred relationships. Species of Maytenus and Gymnosporia from different regions were resolved as polyphyletic groups. Maytenus was resolved in three lineages (New World, African, and Austral-Pacific), while Gymnosporia was resolved in two lineages (New World and Old World). Putterlickia was resolved as nested within the Old World Gymnosporia. Catha edulis (qat, khat) was resolved as sister to the clade of Allocassine, Cassine, Lauridia, and Maurocenia. Gymnosporia cassinoides, which is reportedly chewed as a stimulant in the Canary Islands, was resolved as a derived member of Gymnosporia and is more closely related to Lydenburgia and Putterlickia than it is to Catha. Therefore, all eight of these genera are candidates for containing cathinone- and/or cathine-related alkaloids.

  7. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    Science.gov (United States)

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  8. Concepts and introduction to RNA bioinformatics

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Hofacker, Ivo L.; Ruzzo, Walter L.

    2014-01-01

    RNA bioinformatics and computational RNA biology have emerged from implementing methods for predicting the secondary structure of single sequences. The field has evolved to exploit multiple sequences to take evolutionary information into account, such as compensating (and structure preserving) base...... for interactions between RNA and proteins.Here, we introduce the basic concepts of predicting RNA secondary structure relevant to the further analyses of RNA sequences. We also provide pointers to methods addressing various aspects of RNA bioinformatics and computational RNA biology....

  9. Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

    OpenAIRE

    Zhang, Yunpeng; Liu, Wei; Xu, Yanjun; Li, Chunquan; Wang, Yingying; Yang, Haixiu; Zhang, Chunlong; Su, Fei; Li, Yixue; Li, Xia

    2015-01-01

    Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it i...

  10. Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in Heterosigma akashiwo (Raphidophyceae

    Directory of Open Access Journals (Sweden)

    Jacobs Michael A

    2007-05-01

    Full Text Available Abstract Background Maintenance of homeostasis requires that an organism perceive selected physical and chemical signals within an informationally dense environment. Functionally, an organism uses a variety of signal transduction arrays to amplify and convert these perceived signals into appropriate gene transcriptional responses. These changes in gene expression serve to modify selective metabolic processes and thus optimize reproductive success. Here we analyze a chloroplast-encoded His-to-Asp signal transduction circuit in the stramenopile Heterosigma akashiwo (Hada Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt F.J.R. Taylor]. The presence, structure and putative function of this protein pair are discussed in the context of their evolutionary homologues. Results Bioinformatic analysis of the Heterosigma akashiwo chloroplast genome sequence revealed the presence of a single two-component His-to-Asp (designated Tsg1/Trg1 pair in this stramenopile (golden-brown alga. These data represent the first documentation of a His-to-Asp array in stramenopiles and counter previous reports suggesting that such regulatory proteins are lacking in this taxonomic cluster. Comparison of the 43 kDa H. akashiwo Tsg1 with bacterial sensor kinases showed that the algal protein exhibits a moderately maintained PAS motif in the sensor kinase domain as well as highly conserved H, N, G1 and F motifs within the histidine kinase ATP binding site. Molecular modelling of the 27 kDa H. akashiwo Trg1 regulator protein was consistent with a winged helix-turn-helix identity – a class of proteins that is known to impact gene expression at the level of transcription. The occurrence of Trg1 protein in actively growing H. akashiwo cells was verified by Western analysis. The presence of a PhoB-like RNA polymerase loop in Trg1 and its homologues in the red-algal lineage support the hypothesis that Trg1 and its homologues interact with a sigma 70 (σ70 subunit (encoded by

  11. Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

    International Nuclear Information System (INIS)

    Zhdanov, Vladimir P

    2008-01-01

    The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation

  12. Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation.

    Science.gov (United States)

    Jin, Shuangxia; Singh, Nameirakpam D; Li, Lebin; Zhang, Xianlong; Daniell, Henry

    2015-04-01

    In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  13. Phylogenetic Reconstruction Shows Independent Evolutionary Origins of Mitochondrial Transcription Factors from an Ancient Family of RNA Methyltransferase Proteins.

    Science.gov (United States)

    Aj Harris; Goldman, Aaron David

    2018-04-25

    Here, we generate a robust phylogenetic framework for the rRNA adenine N(6)-methyltransferase (RAMTase) protein family that shows a more ancient and complex evolutionary history within the family than previously reported. RAMTases occur universally by descent across the three domains of life, and typical orthologs within the family perform methylation of the small subunits of ribosomal RNA (rRNA). However, within the RAMTase family, two different groups of mitochondrial transcription factors, mtTFB1 and mtTFB2, have evolved in eukaryotes through neofunctionalization. Previous phylogenetic analyses have suggested that mtTFB1 and mtTFB2 comprise sister clades that arose via gene duplication, which occurred sometime following the endosymbiosis event that produced the mitochondrion. Through dense and taxonomically broad sampling of RAMTase family members especially within bacteria, we found that these eukaryotic mitochondrial transcription factors, mtTFB1 and mtTFB2, have independent origins in phylogenetically distant clades such that their divergence most likely predates the last universal common ancestor of life. The clade of mtTFB2s comprises orthologs in Opisthokonts and the clade of mtTFB1s includes orthologs in Amoebozoa and Metazoa. Thus, we clearly demonstrate that the neofunctionalization producing the transcription factor function evolved twice independently within the RAMTase family. These results are consistent with and help to elucidate outcomes from prior experimental studies, which found that some members of mtTFB1 still perform the ancestral rRNA methylation function, and the results have broader implications for understanding the evolution of new protein functions. Our phylogenetic reconstruction is also in agreement with prior studies showing two independent origins of plastid RAMTases in Viridiplantae and other photosynthetic autotrophs. We believe that this updated phylogeny of RAMTases should provide a robust evolutionary framework for ongoing

  14. 34A, miRNA-944, miRNA-101 and miRNA-218 in cervical cancer

    African Journals Online (AJOL)

    RNAs (21 - 24 nucleotides in length) that are critical for many important processes such as development, ... RNA extraction and reverse transcription. Total RNA was extracted from each of the experimental groups using ... used as an endogenous control to normalize the expression of miRNA-143, miRNA-34A, miRNA-.

  15. Nuclear Export of Messenger RNA

    Directory of Open Access Journals (Sweden)

    Jun Katahira

    2015-03-01

    Full Text Available Transport of messenger RNA (mRNA from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.

  16. RNA viruses in the sea.

    Science.gov (United States)

    Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

    2009-03-01

    Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

  17. Nuclear Export of Messenger RNA

    Science.gov (United States)

    Katahira, Jun

    2015-01-01

    Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex. PMID:25836925

  18. Transfecting Human Monocytes with RNA.

    Science.gov (United States)

    Dannull, Jens; Nair, Smita K

    2016-01-01

    Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

  19. Fast prediction of RNA-RNA interaction using heuristic algorithm.

    Science.gov (United States)

    Montaseri, Soheila

    2015-01-01

    Interaction between two RNA molecules plays a crucial role in many medical and biological processes such as gene expression regulation. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. Some algorithms have been formed to predict the structure of the RNA-RNA interaction. High computational time is a common challenge in most of the presented algorithms. In this context, a heuristic method is introduced to accurately predict the interaction between two RNAs based on minimum free energy (MFE). This algorithm uses a few dot matrices for finding the secondary structure of each RNA and binding sites between two RNAs. Furthermore, a parallel version of this method is presented. We describe the algorithm's concurrency and parallelism for a multicore chip. The proposed algorithm has been performed on some datasets including CopA-CopT, R1inv-R2inv, Tar-Tar*, DIS-DIS, and IncRNA54-RepZ in Escherichia coli bacteria. The method has high validity and efficiency, and it is run in low computational time in comparison to other approaches.

  20. The RNA synthesis machinery of negative-stranded RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  1. The RNA synthesis machinery of negative-stranded RNA viruses

    International Nuclear Information System (INIS)

    Ortín, Juan; Martín-Benito, Jaime

    2015-01-01

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

  2. Generation of miRNA sponge constructs

    NARCIS (Netherlands)

    Kluiver, Joost; Slezak-Prochazka, Izabella; Smigielska-Czepiel, Katarzyna; Halsema, Nancy; Kroesen, Bart-Jan; van den Berg, Anke

    2012-01-01

    MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We

  3. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

    OpenAIRE

    Pinder, Benjamin D; Smibert, Craig A

    2012-01-01

    Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

  4. IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions.

    Science.gov (United States)

    Mann, Martin; Wright, Patrick R; Backofen, Rolf

    2017-07-03

    The IntaRNA algorithm enables fast and accurate prediction of RNA-RNA hybrids by incorporating seed constraints and interaction site accessibility. Here, we introduce IntaRNAv2, which enables enhanced parameterization as well as fully customizable control over the prediction modes and output formats. Based on up to date benchmark data, the enhanced predictive quality is shown and further improvements due to more restrictive seed constraints are highlighted. The extended web interface provides visualizations of the new minimal energy profiles for RNA-RNA interactions. These allow a detailed investigation of interaction alternatives and can reveal potential interaction site multiplicity. IntaRNAv2 is freely available (source and binary), and distributed via the conda package manager. Furthermore, it has been included into the Galaxy workflow framework and its already established web interface enables ad hoc usage. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. A model for tetrapyrrole synthesis as the primary mechanism for plastid-to-nucleus signaling during chloroplast biogenesis

    Directory of Open Access Journals (Sweden)

    Matthew J. Terry

    2013-02-01

    Full Text Available Chloroplast biogenesis involves the co-ordinated expression of the chloroplast and nuclear genomes, requiring information to be sent from the developing chloroplasts to the nucleus. This is achieved through retrograde signaling pathways and can be demonstrated experimentally using the photobleaching herbicide, Norflurazon, which results in chloroplast damage and the reduced expression of many photosynthesis-related, nuclear genes in seedlings. Genetic analysis of this pathway points to a major role for tetrapyrrole synthesis in retrograde signaling, as well as a strong interaction with light-signaling pathways. Currently, the best model to explain the genetic data is that a specific heme pool generated by flux through ferrochelatase-1 functions as a positive signal to promote the expression of genes required for chloroplast development. We propose that this heme-related signal is the primary positive signal during chloroplast biogenesis, and that treatments and mutations affecting chloroplast transcription, RNA editing, translation, or protein import all impact on the synthesis and/or processing of this signal. A positive signal is consistent with the need to provide information on chloroplast status at all times. We further propose that GUN1 normally serves to restrict the production of the heme signal. In addition to a positive signal re-enforcing chloroplast development under normal conditions, aberrant chloroplast development may produce a negative signal due to accumulation of unbound chlorophyll biosynthesis intermediates, such as Mg-porphyrins. Under these conditions a rapid shut-down of tetrapyrrole synthesis is required. We propose that accumulation of these intermediates results in a rapid light-dependent inhibition of nuclear gene expression that is most likely mediated via singlet oxygen generated by photo-excitation of Mg-porphyrins. Thus, the tetrapyrrole pathway may provide both positive and inhibitory signals to control

  6. Direct, rapid RNA sequence analysis

    International Nuclear Information System (INIS)

    Peattie, D.A.

    1987-01-01

    The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

  7. Cofactors in the RNA World

    Science.gov (United States)

    Ditzler, Mark A.

    2014-01-01

    RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

  8. Efficient RNA structure comparison algorithms.

    Science.gov (United States)

    Arslan, Abdullah N; Anandan, Jithendar; Fry, Eric; Monschke, Keith; Ganneboina, Nitin; Bowerman, Jason

    2017-12-01

    Recently proposed relative addressing-based ([Formula: see text]) RNA secondary structure representation has important features by which an RNA structure database can be stored into a suffix array. A fast substructure search algorithm has been proposed based on binary search on this suffix array. Using this substructure search algorithm, we present a fast algorithm that finds the largest common substructure of given multiple RNA structures in [Formula: see text] format. The multiple RNA structure comparison problem is NP-hard in its general formulation. We introduced a new problem for comparing multiple RNA structures. This problem has more strict similarity definition and objective, and we propose an algorithm that solves this problem efficiently. We also develop another comparison algorithm that iteratively calls this algorithm to locate nonoverlapping large common substructures in compared RNAs. With the new resulting tools, we improved the RNASSAC website (linked from http://faculty.tamuc.edu/aarslan ). This website now also includes two drawing tools: one specialized for preparing RNA substructures that can be used as input by the search tool, and another one for automatically drawing the entire RNA structure from a given structure sequence.

  9. Alternative RNA splicing and cancer

    Science.gov (United States)

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  10. The ViennaRNA web services.

    Science.gov (United States)

    Gruber, Andreas R; Bernhart, Stephan H; Lorenz, Ronny

    2015-01-01

    The ViennaRNA package is a widely used collection of programs for thermodynamic RNA secondary structure prediction. Over the years, many additional tools have been developed building on the core programs of the package to also address issues related to noncoding RNA detection, RNA folding kinetics, or efficient sequence design considering RNA-RNA hybridizations. The ViennaRNA web services provide easy and user-friendly web access to these tools. This chapter describes how to use this online platform to perform tasks such as prediction of minimum free energy structures, prediction of RNA-RNA hybrids, or noncoding RNA detection. The ViennaRNA web services can be used free of charge and can be accessed via http://rna.tbi.univie.ac.at.

  11. Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

    NARCIS (Netherlands)

    Kluiver, Joost; Gibcus, Johan H.; Hettinga, Chris; Adema, Annelies; Richter, Mareike K. S.; Halsema, Nancy; Slezak-Prochazka, Izabella; Ding, Ye; Kroesen, Bart-Jan; van den Berg, Anke

    2012-01-01

    MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and

  12. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    Science.gov (United States)

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants.

    Science.gov (United States)

    Zang, Xinshan; Geng, Xiaoli; Liu, Kelu; Wang, Fei; Liu, Zhenshan; Zhang, Liyuan; Zhao, Yue; Tian, Xuejun; Hu, Zhaorong; Yao, Yingyin; Ni, Zhongfu; Xin, Mingming; Sun, Qixin; Peng, Huiru

    2017-05-01

    Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Allopatric speciation despite historical gene flow: Divergence and hybridization in Carex furva and C. lucennoiberica (Cyperaceae) inferred from plastid and nuclear RAD-seq data.

    Science.gov (United States)

    Maguilla, Enrique; Escudero, Marcial; Hipp, Andrew L; Luceño, Modesto

    2017-10-01

    Gene flow among incipient species can act as a creative or destructive force in the speciation process, generating variation on which natural selection can act while, potentially, undermining population divergence. The flowering plant genus Carex exhibits a rapid and relatively recent radiation with many species limits still unclear. This is the case with the Iberian Peninsula (Spain and Portugal)-endemic C. lucennoiberica, which lay unrecognized within Carex furva until its recent description as a new species. In this study, we test how these species were impacted by interspecific gene flow during speciation. We sampled the full range of distribution of C. furva (15 individuals sampled) and C. lucennoiberica (88 individuals), sequenced two cpDNA regions (atpI-atpH, psbA-trnH) and performed genomic sequencing of 45,100 SNPs using restriction site-associated DNA sequencing (RAD-seq). We utilized a set of partitioned D-statistic tests and demographic analyses to study the degree and direction of introgression. Additionally, we modelled species distributions to reconstruct changes in range distribution during glacial and interglacial periods. Plastid, nuclear and morphological data strongly support divergence between species with subsequent gene flow. Combined with species distribution modelling, these data support a scenario of allopatry leading to species divergence, followed by secondary contact and gene flow due to long-distance dispersal and/or range expansions and contractions in response to Quaternary glacial cycles. We conclude that this is a case of allopatric speciation despite historical secondary contacts, which could have temporally influenced the speciation process, contributing to the knowledge of forces that are driving or counteracting speciation. © 2017 John Wiley & Sons Ltd.

  15. Allotetraploid origin and divergence in Eleusine (Chloridoideae, Poaceae): evidence from low-copy nuclear gene phylogenies and a plastid gene chronogram.

    Science.gov (United States)

    Liu, Qing; Triplett, Jimmy K; Wen, Jun; Peterson, Paul M

    2011-11-01

    Eleusine (Poaceae) is a small genus of the subfamily Chloridoideae exhibiting considerable morphological and ecological diversity in East Africa and the Americas. The interspecific phylogenetic relationships of Eleusine are investigated in order to identify its allotetraploid origin, and a chronogram is estimated to infer temporal relationships between palaeoenvironment changes and divergence of Eleusine in East Africa. Two low-copy nuclear (LCN) markers, Pepc4 and EF-1α, were analysed using parsimony, likelihood and Bayesian approaches. A chronogram of Eleusine was inferred from a combined data set of six plastid DNA markers (ndhA intron, ndhF, rps16-trnK, rps16 intron, rps3, and rpl32-trnL) using the Bayesian dating method. The monophyly of Eleusine is strongly supported by sequence data from two LCN markers. In the cpDNA phylogeny, three tetraploid species (E. africana, E. coracana and E. kigeziensis) share a common ancestor with the E. indica-E. tristachya clade, which is considered a source of maternal parents for allotetraploids. Two homoeologous loci are isolated from three tetraploid species in the Pepc4 phylogeny, and the maternal parents receive further support. The A-type EF-1α sequences possess three characters, i.e. a large number of variations of intron 2; clade E-A distantly diverged from clade E-B and other diploid species; and seven deletions in intron 2, implying a possible derivation through a gene duplication event. The crown age of Eleusine and the allotetraploid lineage are 3·89 million years ago (mya) and 1·40 mya, respectively. The molecular data support independent allotetraploid origins for E. kigeziensis and the E. africana-E. coracana clade. Both events may have involved diploids E. indica and E. tristachya as the maternal parents, but the paternal parents remain unidentified. The habitat-specific hypothesis is proposed to explain the divergence of Eleusine and its allotetraploid lineage.

  16. A plastid phylogeny and character evolution of the Old World fern genus Pyrrosia (Polypodiaceae) with the description of a new genus: Hovenkampia (Polypodiaceae).

    Science.gov (United States)

    Zhou, Xin-Mao; Zhang, Liang; Chen, Cheng-Wei; Li, Chun-Xiang; Huang, Yao-Moan; Chen, De-Kui; Lu, Ngan Thi; Cicuzza, Daniele; Knapp, Ralf; Luong, Thien Tam; Nitta, Joel H; Gao, Xin-Fen; Zhang, Li-Bing

    2017-09-01

    The Old World fern genus Pyrrosia (Polypodiaceae) offers a rare system in ferns to study morphological evolution because almost all species of this genus are well studied for their morphology, anatomy, and spore features, and various hypotheses have been proposed in terms of the phylogeny and evolution in this genus. However, the molecular phylogeny of the genus lags behind. The monophyly of the genus has been uncertain and a modern phylogenetic study of the genus based on molecular data has been lacking. In the present study, DNA sequences of five plastid markers of 220 accessions of Polypodiaceae representing two species of Drymoglossum, 14 species of Platycerium, 50 species of Pyrrosia, and the only species of Saxiglossum (subfamily Platycerioideae), and 12 species of other Polypodiaceae representing the remaining four subfamilies are used to infer a phylogeny of the genus. Major results and conclusions of this study include: (1) Pyrrosia as currently circumscribed is paraphyletic in relation to Platycerium and can be divided into two genera: Pyrrosia s.s. and Hovenkampia (gen. nov.), with Hovenkampia and Platycerium forming a strongly supported clade sister to Pyrrosia s.s.; (2) Subfamily Platycerioideae should contain three genera only, Hovenkampia, Platycerium, and Pyrrosia s.s.; (3) Based on the molecular phylogeny, macromorphology, anatomical features, and spore morphology, four major clades in the genus are identified and three of the four are further resolved into four, four, and six subclades, respectively; (4) Three species, P. angustissima, P. foveolata, and P. mannii, not assigned to any groups by Hovenkamp (1986) because of their unusual morphology, each form monospecific clades; (5) Drymoglossum is not monophyletic and those species previously assigned to this genus are resolved in two different subclades; (6) Saxiglossum is resolved as the first lineage in the Niphopsis clade; and (7) The evolution of ten major morphological characters in the

  17. Identification of Subtype Specific miRNA-mRNA Functional Regulatory Modules in Matched miRNA-mRNA Expression Data: Multiple Myeloma as a Case

    Directory of Open Access Journals (Sweden)

    Yunpeng Zhang

    2015-01-01

    Full Text Available Identification of miRNA-mRNA modules is an important step to elucidate their combinatorial effect on the pathogenesis and mechanisms underlying complex diseases. Current identification methods primarily are based upon miRNA-target information and matched miRNA and mRNA expression profiles. However, for heterogeneous diseases, the miRNA-mRNA regulatory mechanisms may differ between subtypes, leading to differences in clinical behavior. In order to explore the pathogenesis of each subtype, it is important to identify subtype specific miRNA-mRNA modules. In this study, we integrated the Ping-Pong algorithm and multiobjective genetic algorithm to identify subtype specific miRNA-mRNA functional regulatory modules (MFRMs through integrative analysis of three biological data sets: GO biological processes, miRNA target information, and matched miRNA and mRNA expression data. We applied our method on a heterogeneous disease, multiple myeloma (MM, to identify MM subtype specific MFRMs. The constructed miRNA-mRNA regulatory networks provide modular outlook at subtype specific miRNA-mRNA interactions. Furthermore, clustering analysis demonstrated that heterogeneous MFRMs were able to separate corresponding MM subtypes. These subtype specific MFRMs may aid in the further elucidation of the pathogenesis of each subtype and may serve to guide MM subtype diagnosis and treatment.

  18. Novel 9-cis/all-trans β-carotene isomerases from plastidic oil bodies in Dunaliella bardawil catalyze the conversion of all-trans to 9-cis β-carotene.

    Science.gov (United States)

    Davidi, Lital; Pick, Uri

    2017-06-01

    We identified and demonstrated the function of 9-cis/all-trans β-carotene isomerases in plastidic globules of Dunaliella bardawil, the species accumulating the highest levels of 9-cis β-carotene that is essential for humans. The halotolerant alga Dunaliella bardawil is unique in that it accumulates under light stress high levels of β-carotene in plastidic lipid globules. The pigment is composed of two major isomers: all-trans β-carotene, the common natural form of this pigment, and 9-cis β-carotene. The biosynthetic pathway of β-carotene is known, but it is not clear how the 9-cis isomer is formed. We identified in plastidic lipid globules that were isolated from D. bardawil two proteins with high sequence homology to the D27 protein-a 9-cis/all-trans β-carotene isomerase from rice (Alder et al. Science 335:1348-1351, 2012). The proteins are enriched in the oil globules by 6- to 17-fold compared to chloroplast proteins. The expression of the corresponding genes, 9-cis-βC-iso1 and 9-cis-βC-iso2, is enhanced under light stress. The synthetic proteins catalyze in vitro conversion of all-trans to 9-cis β-carotene. Expression of the 9-cis-βC-iso1 or of 9-cis-βC-iso2 genes in an E. coli mutant line that harbors β-carotene biosynthesis genes enhanced the conversion of all-trans into 9-cis β-carotene. These results suggest that 9-cis-βC-ISO1 and 9-cis-βC-ISO2 proteins are responsible for the formation of 9-cis β-carotene in D. bardawil under stress conditions.

  19. The largest subunit of RNA polymerase II from the Glaucocystophyta: functional constraint and short-branch exclusion in deep eukaryotic phylogeny

    Directory of Open Access Journals (Sweden)

    Stiller John W

    2005-12-01

    Full Text Available Abstract Background Evolutionary analyses of the largest subunit of RNA polymerase II (RPB1 have yielded important and at times provocative results. One particularly troublesome outcome is the consistent inference of independent origins of red algae and green plants, at odds with the more widely accepted view of a monophyletic Plantae comprising all eukaryotes with primary plastids. If the hypothesis of a broader kingdom Plantae is correct, then RPB1 trees likely reflect a persistent phylogenetic artifact. To gain a better understanding of RNAP II evolution, and the presumed artifact relating to green plants and red algae, we isolated and analyzed RPB1 from representatives of Glaucocystophyta, the third eukaryotic group with primary plastids. Results Phylogenetic analyses incorporating glaucocystophytes do not recover a monophyletic Plantae; rather they result in additional conflicts with the most widely held views on eukaryotic relationships. In particular, glaucocystophytes are recovered as sister to several amoebozoans with strong support. A detailed investigation shows that this clade can be explained by what we call "short-branch exclusion," a phylogenetic artifact integrally associated with "long-branch attraction." Other systematic discrepancies observed in RPB1 trees can be explained as phylogenetic artifacts; however, these apparent artifacts also appear in regions of the tree that support widely held views of eukaryotic evolution. In fact, most of the RPB1 tree is consistent with artifacts of rate variation among sequences and co-variation due to functional constraints related to C-terminal domain based RNAP II transcription. Conclusion Our results reveal how subtle and easily overlooked biases can dominate the overall results of molecular phylogenetic analyses of ancient eukaryotic relationships. Sources of potential phylogenetic artifact should be investigated routinely, not just when obvious "long-branch attraction" is encountered.

  20. Predicting and Modeling RNA Architecture

    Science.gov (United States)

    Westhof, Eric; Masquida, Benoît; Jossinet, Fabrice

    2011-01-01

    SUMMARY A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values. PMID:20504963

  1. Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

    Science.gov (United States)

    Ranade, Sonali Sachin; García-Gil, María Rosario; Rosselló, Josep A

    2016-04-01

    Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies). This study aims to trace the ndh genes in the nuclear and organellar Norway spruce genomes. The plastid genomes of higher plants contain 11 ndh genes which are homologues of mitochondrial genes encoding subunits of the proton-pumping NADH-dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) or complex I (electron transport chain). Ndh genes encode 11 NDH polypeptides forming the Ndh complex (analogous to complex I) which seems to be primarily involved in chloro-respiration processes. We considered ndh genes from the plastidial genome of four gymnosperms (Cryptomeria japonica, Cycas revoluta, Ginkgo biloba, Podocarpus totara) and a single angiosperm species (Arabidopsis thaliana) to trace putative homologs in the nuclear and organellar Norway spruce genomes using tBLASTn to assess the evolutionary fate of ndh genes in Norway spruce and to address their genomic location(s), structure, integrity and functionality. The results obtained from tBLASTn were subsequently analyzed by performing homology search for finding ndh specific conserved domains using conserved domain search. We report the presence of non-functional plastid ndh gene fragments, excepting ndhE and ndhG genes, in the nuclear genome of Norway spruce. Regulatory transcriptional elements like promoters, TATA boxes and enhancers were detected in the upstream regions of some ndh fragments. We also found transposable elements in the flanking regions of few ndh fragments suggesting nuclear rearrangements in those regions. These evidences

  2. Shielding the messenger (RNA): microRNA-based anticancer therapies

    Science.gov (United States)

    Sotillo, Elena; Thomas-Tikhonenko, Andrei

    2011-01-01

    It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21–22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded. PMID:21514318

  3. Chaperoning 5S RNA assembly.

    Science.gov (United States)

    Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

    2015-07-01

    In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. © 2015 Madru et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hiraku Takada

    Full Text Available Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon, within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons

  5. RegRNA: an integrated web server for identifying regulatory RNA motifs and elements

    OpenAIRE

    Huang, Hsi-Yuan; Chien, Chia-Hung; Jen, Kuan-Hua; Huang, Hsien-Da

    2006-01-01

    Numerous regulatory structural motifs have been identified as playing essential roles in transcriptional and post-transcriptional regulation of gene expression. RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5′-untra...

  6. Analysis of intermolecular RNA-RNA recombination by rubella virus

    International Nuclear Information System (INIS)

    Adams, Sandra D.; Tzeng, W.-P.; Chen, M.-H.; Frey, Teryl K.

    2003-01-01

    To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating

  7. MicroRNA mimicry blocks pulmonary fibrosis

    NARCIS (Netherlands)

    Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular

  8. Biochemistry and Function of the RNA Exosomes

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Chlebowski, Aleksander; Dziembowski, Andrzej

    2012-01-01

    Discovery of the evolutionary conserved RNA exosome was a milestone in RNA biology. First identified as an activity essential for the processing of ribosomal RNA, the exosome has since proved to be central for RNA processing and degradation in both the nucleus and the cytoplasm of eukaryotic cell...

  9. The crystal structure of tRNA

    Indian Academy of Sciences (India)

    Madhu

    of yeast alanine tRNA by Robert Holley's group at Cornell. University ... decode nonsense codons) with John Smith and Brenner. However, my ... tRNA from 10 g of unfractionated tRNA. ... tRNA crystals were, in fact, protein (Hendrikson et al.

  10. A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

    Directory of Open Access Journals (Sweden)

    Baodong Wu

    2009-03-01

    Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

  11. Glia to axon RNA transfer.

    Science.gov (United States)

    Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

    2014-03-01

    The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased. Copyright © 2013 Wiley Periodicals, Inc.

  12. On topological RNA interaction structures.

    Science.gov (United States)

    Qin, Jing; Reidys, Christian M

    2013-07-01

    Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

  13. Tapping the RNA world for therapeutics.

    Science.gov (United States)

    Lieberman, Judy

    2018-04-16

    A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

  14. RNA-Based Vaccines in Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Megan A. McNamara

    2015-01-01

    Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

  15. A Regulatory RNA Inducing Transgenerationally Inherited Phenotypes

    DEFF Research Database (Denmark)

    Jensen, Lea Møller

    . The variation in Arabidopsis enables different regulatory networks and mechanisms to shape the phenotypic characteristics. The thesis describes the identification of regulatory RNA encoded by an enzyme encoding gene. The RNA regulates by inducing transgenerationally inherited phenotypes. The function of the RNA...... is dependent on the genetic background illustrating that polymorphisms are found in either interactors or target genes of the RNA. Furthermore, the RNA provides a mechanistic link between accumulation of glucosinolate and onset of flowering....

  16. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form......Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...

  17. TargetRNA: a tool for predicting targets of small RNA action in bacteria

    OpenAIRE

    Tjaden, Brian

    2008-01-01

    Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

  18. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

    Previously, an RNA-dependent RNA polymerase produced upon infection of

  19. RNA Study Using DNA Nanotechnology.

    Science.gov (United States)

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    DEFF Research Database (Denmark)

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

    2007-01-01

    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  1. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

    Science.gov (United States)

    Pinder, Benjamin D; Smibert, Craig A

    2013-01-01

    Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

  2. Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase.

    Science.gov (United States)

    Heck, Julie A; Meng, Xiao; Frick, David N

    2009-04-01

    Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111-22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

  3. Role of CBCA in RNA biogenesis

    DEFF Research Database (Denmark)

    Iasillo, Claudia

    RNA transcription and RNA processing are key steps in eukaryotic gene expression, which includes, therefore, RNA synthesis by RNA polymerase enzymes and a range of modifications of the pre-mRNA before the transcript can leave the nucleus and reach the cytoplasm for translation. Interestingly......, a large body of evidence suggests that these RNA processing events occur often already during transcription. One of these modifications, the co-transcriptional 5’ end capping of a nascent RNA, is occurring specifically during RNA polymerase II (RNAPII) transcription. The 5’ cap exerts its role via...... the nuclear Cap Binding Complex (CBC). This thesis focuses on the protein ARS2, which binds the CBC to form the CBCA complex. CBCA can further associate with different proteins playing different roles in RNA metabolism. For example, CBCA binds the Nuclear Exosome Targeting Complex (NEXT), which...

  4. Hydration dependent dynamics in RNA

    International Nuclear Information System (INIS)

    Olsen, Greg L.; Bardaro, Michael F.; Echodu, Dorothy C.; Drobny, Gary P.; Varani, Gabriele

    2009-01-01

    The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in 2 H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state 13 C relaxation measurements, we establish that ns-μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration

  5. RNA Editing in Plant Mitochondria

    Science.gov (United States)

    Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

    1989-12-01

    Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

  6. Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

    OpenAIRE

    Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

    2003-01-01

    For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

  7. Modular arrangement of regulatory RNA elements.

    Science.gov (United States)

    Roßmanith, Johanna; Narberhaus, Franz

    2017-03-04

    Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.

  8. MicroRNA Delivery for Regenerative Medicine

    OpenAIRE

    Peng, Bo; Chen, Yongming; Leong, Kam W.

    2015-01-01

    MicroRNA (miRNA) directs post-transcriptional regulation of a network of genes by targeting mRNA. Although relatively recent in development, many miRNAs direct differentiation of various stem cells including induced pluripotent stem cells (iPSCs), a major player in regenerative medicine. An effective and safe delivery of miRNA holds the key to translating miRNA technologies. Both viral and nonviral delivery systems have seen success in miRNA delivery, and each approach possesses advantages an...

  9. Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

    International Nuclear Information System (INIS)

    Kolakofsky, Daniel; Le Mercier, Philippe; Iseni, Frederic; Garcin, Dominique

    2004-01-01

    mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

  10. iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

    Directory of Open Access Journals (Sweden)

    Jittrawan Thaiprasit

    2016-03-01

    Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

  11. Comprehensive characterization of lncRNA-mRNA related ceRNA network across 12 major cancers

    Science.gov (United States)

    Feng, Li; Li, Feng; Sun, Zeguo; Wu, Tan; Shi, Xinrui; Li, Jing; Li, Xia

    2016-01-01

    Recent studies indicate that long noncoding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNAs) to indirectly regulate mRNAs through shared microRNAs, which represents a novel layer of RNA crosstalk and plays critical roles in the development of tumor. However, the global regulation landscape and characterization of these lncRNA related ceRNA crosstalk in cancers is still largely unknown. Here, we systematically characterized the lncRNA related ceRNA interactions across 12 major cancers and the normal physiological states by integrating multidimensional molecule profiles of more than 5000 samples. Our study suggest the large difference of ceRNA regulation between normal and tumor states and the higher similarity across similar tissue origin of tumors. The ceRNA related molecules have more conserved features in tumor networks and they play critical roles in both the normal and tumorigenesis processes. Besides, lncRNAs in the pan-cancer ceRNA network may be potential biomarkers of tumor. By exploring hub lncRNAs, we found that these conserved key lncRNAs dominate variable tumor hallmark processes across pan-cancers. Network dynamic analysis highlights the critical roles of ceRNA regulation in tumorigenesis. By analyzing conserved ceRNA interactions, we found that miRNA mediate ceRNA regulation showed different patterns across pan-cancer; while analyzing the cancer specific ceRNA interactions reveal that lncRNAs synergistically regulated tumor driver genes of cancer hallmarks. Finally, we found that ceRNA modules have the potential to predict patient survival. Overall, our study systematically dissected the lncRNA related ceRNA networks in pan-cancer that shed new light on understanding the molecular mechanism of tumorigenesis. PMID:27580177

  12. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

    Directory of Open Access Journals (Sweden)

    Darrin V. Bann

    2012-06-01

    Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

  13. How the RNA isolation method can affect microRNA microarray results

    DEFF Research Database (Denmark)

    Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

    2011-01-01

    RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

  14. Topology and prediction of RNA pseudoknots

    DEFF Research Database (Denmark)

    Reidys, Christian; Huang, Fenix; Andersen, Jørgen Ellegaard

    2011-01-01

    Motivation: Several dynamic programming algorithms for predicting RNA structures with pseudoknots have been proposed that differ dramatically from one another in the classes of structures considered. Results: Here, we use the natural topological classification of RNA structures in terms...

  15. Terpenoid Metabolism in Plastids 1

    Science.gov (United States)

    Camara, Bilal; Bardat, Françoise; Seye, Ababacar; D'Harlingue, Alain; Monéger, René

    1982-01-01

    The synthesis of α-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l-methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:γ-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction. PMID:16662717

  16. RNA Structural Alignments, Part I

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Gorodkin, Jan

    2014-01-01

    Simultaneous alignment and secondary structure prediction of RNA sequences is often referred to as "RNA structural alignment." A class of the methods for structural alignment is based on the principles proposed by Sankoff more than 25 years ago. The Sankoff algorithm simultaneously folds and aligns...... is so high that it took more than a decade before the first implementation of a Sankoff style algorithm was published. However, with the faster computers available today and the improved heuristics used in the implementations the Sankoff-based methods have become practical. This chapter describes...... the methods based on the Sankoff algorithm. All the practical implementations of the algorithm use heuristics to make them run in reasonable time and memory. These heuristics are also described in this chapter....

  17. Fatgraph models of RNA structure

    Directory of Open Access Journals (Sweden)

    Huang Fenix

    2017-01-01

    Full Text Available In this review paper we discuss fatgraphs as a conceptual framework for RNA structures. We discuss various notions of coarse-grained RNA structures and relate them to fatgraphs.We motivate and discuss the main intuition behind the fatgraph model and showcase its applicability to canonical as well as noncanonical base pairs. Recent discoveries regarding novel recursions of pseudoknotted (pk configurations as well as their translation into context-free grammars for pk-structures are discussed. This is shown to allow for extending the concept of partition functions of sequences w.r.t. a fixed structure having non-crossing arcs to pk-structures. We discuss minimum free energy folding of pk-structures and combine these above results outlining how to obtain an inverse folding algorithm for PK structures.

  18. RNA

    African Journals Online (AJOL)

    SARAH

    30 nov. 2013 ... Keywords: FMNR, mode of management, re-greening, leadership, evolutionary trend. INTRODUCTION .... régénération : L'évolution de la densité des ligneux entre. 2005 et 2012 ..... la production et la qualité fourragères de la.

  19. Nonradioactive RNA mobility shift with chemiluminescent detection ...

    African Journals Online (AJOL)

    hesham

    RNA mobility shift is one among many procedures used to study RNA-protein interaction. Yet, there are some limitations for the radioactive RNA mobility shift including; 1) the risk of using radiolabeled nucleotides, 2) the long time to get the results; this could range from days to weeks, and 3) its high cost as compared to ...

  20. Optimization of chemiluminescent detection of mitochondrial RNA ...

    African Journals Online (AJOL)

    RNA mobility shift is one among many procedures used to study RNA-protein interaction. Yet, there are some limitations for the radioactive RNA mobility shift including; 1) the risk of using radiolabeled nucleotides, 2) the long time to get the results; this could range from days to weeks, and 3) its high cost as compared to ...

  1. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  2. Analysis of RNA metabolism in fission yeast

    DEFF Research Database (Denmark)

    Wise, Jo Ann; Nielsen, Olaf

    2017-01-01

    Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

  3. Tospovirus : induction and suppression of RNA silencing

    NARCIS (Netherlands)

    Hedil, Marcio

    2016-01-01

    While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants,

  4. A Specific Hepatic Transfer RNA for Phosphoserine*

    Science.gov (United States)

    Mäenpää, Pekka H.; Bernfield, Merton R.

    1970-01-01

    Radioactive O-phosphoryl-L-serine was detected after alkaline deacylation of rat and rooster liver [3H]seryl-tRNA acylated in vitro with homologous synthetases. Ribonuclease treatment of this tRNA yielded a compound with the properties of phosphoseryl-adenosine. Benzoylated DEAE-cellulose chromatography of seryl-tRNA yielded four distinct peaks, only one of which contained phosphoserine. A unique fraction for phosphoserine was also found on chromatography of nonacylated tRNA. In ribosome binding studies, this fraction responded very slightly with poly(U,C), but not with any of the known serine trinucleotide codons. Substantial incorporation of [3H]-serine into protein from this tRNA species was observed in an aminoacyl-tRNA dependent polysomal system derived from chick oviducts. No phosphoserine was found in Escherichia coli or yeast seryl-tRNA acylated with homologous enzymes, nor in E. coli seryl-tRNA acylated with liver synthetase. In the absence of tRNA, free phosphoserine was not formed in reaction mixtures, which suggests that phosphoseryl-tRNA arises by phosphorylation of the unique seryl-tRNA species. These results demonstrate a discrete tRNASer species in rat and rooster liver containing phosphoserine and suggest that this tRNA is involved in ribosomal polypeptide synthesis. PMID:4943179

  5. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  6. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  7. Supplementary data: Materials and methods RNA expression ...

    Indian Academy of Sciences (India)

    ritt8

    Supplementary data: Materials and methods. RNA expression analysis. Freshly collected tissue was taken in TRIzol reagent for total RNA isolation according to the manufacturer's protocol. The cDNA synthesis was carried out in 1 μg total RNA using Random hexamer (Invitrogen, Carlsbad, USA) and Superscript III ...

  8. Regulatory RNAs derived from transfer RNA?

    Science.gov (United States)

    Pederson, Thoru

    2010-10-01

    Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

  9. Regulatory BC1 RNA in Cognitive Control

    Science.gov (United States)

    Iacoangeli, Anna; Dosunmu, Aderemi; Eom, Taesun; Stefanov, Dimitre G.; Tiedge, Henri

    2017-01-01

    Dendritic regulatory BC1 RNA is a non-protein-coding (npc) RNA that operates in the translational control of gene expression. The absence of BC1 RNA in BC1 knockout (KO) animals causes translational dysregulation that entails neuronal phenotypic alterations including prolonged epileptiform discharges, audiogenic seizure activity in vivo, and…

  10. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6

    DEFF Research Database (Denmark)

    Devert, Anthony; Fabre, Nicolas; Floris, Maina Huguette Joséphine

    2015-01-01

    ) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer......Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA......-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer...

  11. Effective Anti-miRNA Oligonucleotides Show High Releasing Rate of MicroRNA from RNA-Induced Silencing Complex.

    Science.gov (United States)

    Ariyoshi, Jumpei; Matsuyama, Yohei; Kobori, Akio; Murakami, Akira; Sugiyama, Hiroshi; Yamayoshi, Asako

    2017-10-01

    MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC 50 ) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC 50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.

  12. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

    Science.gov (United States)

    Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

    2010-11-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

  13. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  14. The early history of tRNA recognition by aminoacyl-tRNA synthetases

    Indian Academy of Sciences (India)

    Madhu

    2006-10-04

    Oct 4, 2006 ... Discovery of aminoacyl-tRNA synthetases and importance ... The pioneering work of Fritz Lipmann on the high-energy ... the peculiar structural and functional relationships tRNAs ... a bulk of only 20 families of tRNA molecules in contrast ...... balance of tRNA and aminoacyl-tRNA synthetase; Science 242.

  15. Cooperation of an RNA Packaging Signal and a Viral Envelope Protein in Coronavirus RNA Packaging

    OpenAIRE

    Narayanan, Krishna; Makino, Shinji

    2001-01-01

    Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA packaging signal is necessary and suffi...

  16. Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

    Science.gov (United States)

    Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

    2006-01-01

    RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

  17. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  18. Using RNA Interference to Study Protein Function

    OpenAIRE

    Curtis, Carol D.; Nardulli, Ann M.

    2009-01-01

    RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the effect of a putative regulatory protein on gene expression can be delineated. Methods of assessing the effectiveness of the siRNA procedure using real time quantitative PCR and Western analysis are also in...

  19. Analysis of extracellular RNA by digital PCR

    Directory of Open Access Journals (Sweden)

    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  20. Characteristics and Prediction of RNA Structure

    Directory of Open Access Journals (Sweden)

    Hengwu Li

    2014-01-01

    Full Text Available RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, and L, where L is the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding.

  1. siRNA and innate immunity.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; MacLachlan, Ian

    2009-06-01

    Canonical small interfering RNA (siRNA) duplexes are potent activators of the mammalian innate immune system. The induction of innate immunity by siRNA is dependent on siRNA structure and sequence, method of delivery, and cell type. Synthetic siRNA in delivery vehicles that facilitate cellular uptake can induce high levels of inflammatory cytokines and interferons after systemic administration in mammals and in primary human blood cell cultures. This activation is predominantly mediated by immune cells, normally via a Toll-like receptor (TLR) pathway. The siRNA sequence dependency of these pathways varies with the type and location of the TLR involved. Alternatively nonimmune cell activation may also occur, typically resulting from siRNA interaction with cytoplasmic RNA sensors such as RIG1. As immune activation by siRNA-based drugs represents an undesirable side effect due to the considerable toxicities associated with excessive cytokine release in humans, understanding and abrogating this activity will be a critical component in the development of safe and effective therapeutics. This review describes the intracellular mechanisms of innate immune activation by siRNA, the design of appropriate sequences and chemical modification approaches, and suitable experimental methods for studying their effects, with a view toward reducing siRNA-mediated off-target effects.

  2. TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

    Science.gov (United States)

    Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

  3. MysiRNA-designer: a workflow for efficient siRNA design.

    Directory of Open Access Journals (Sweden)

    Mohamed Mysara

    Full Text Available The design of small interfering RNA (siRNA is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.

  4. 5S rRNA and ribosome.

    Science.gov (United States)

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  5. Kin Selection in the RNA World.

    Science.gov (United States)

    Levin, Samuel R; West, Stuart A

    2017-12-05

    Various steps in the RNA world required cooperation. Why did life's first inhabitants, from polymerases to synthetases, cooperate? We develop kin selection models of the RNA world to answer these questions. We develop a very simple model of RNA cooperation and then elaborate it to model three relevant issues in RNA biology: (1) whether cooperative RNAs receive the benefits of cooperation; (2) the scale of competition in RNA populations; and (3) explicit replicator diffusion and survival. We show: (1) that RNAs are likely to express partial cooperation; (2) that RNAs will need mechanisms for overcoming local competition; and (3) in a specific example of RNA cooperation, persistence after replication and offspring diffusion allow for cooperation to overcome competition. More generally, we show how kin selection can unify previously disparate answers to the question of RNA world cooperation.

  6. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    Directory of Open Access Journals (Sweden)

    Ching-Ping Lin

    Full Text Available We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911. In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum, two monocots (Oryza sativa and Zea mays, two gymnosperms (Pinus taeda and Ginkgo biloba and one moss (Physcomitrella patens. Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

  7. MicroRNA and cancer

    DEFF Research Database (Denmark)

    Jansson, Martin D; Lund, Anders H

    2012-01-01

    biological phenomena and pathologies. The best characterized non-coding RNA family consists in humans of about 1400 microRNAs for which abundant evidence have demonstrated fundamental importance in normal development, differentiation, growth control and in human diseases such as cancer. In this review, we...... summarize the current knowledge and concepts concerning the involvement of microRNAs in cancer, which have emerged from the study of cell culture and animal model systems, including the regulation of key cancer-related pathways, such as cell cycle control and the DNA damage response. Importantly, micro...

  8. RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

    Science.gov (United States)

    Moss, Walter N

    2018-03-01

    With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

    Science.gov (United States)

    Haque, Farzin; Pi, Fengmei; Zhao, Zhengyi; Gu, Shanqing; Hu, Haibo; Yu, Hang; Guo, Peixuan

    2018-01-01

    In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs. © 2017 Wiley Periodicals, Inc.

  10. Evaluation of microRNA alignment techniques

    Science.gov (United States)

    Kaspi, Antony; El-Osta, Assam

    2016-01-01

    Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

  11. MicroRNA mimicry blocks pulmonary fibrosis

    Science.gov (United States)

    Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis. PMID:25239947

  12. Modulation of RNA function by aminoglycoside antibiotics.

    Science.gov (United States)

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-04

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

  13. Movement of regulatory RNA between animal cells.

    Science.gov (United States)

    Jose, Antony M

    2015-07-01

    Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions. © 2015 Wiley Periodicals, Inc.

  14. Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

    Science.gov (United States)

    Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

    2017-01-01

    microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

  15. Henipavirus RNA in African bats.

    Directory of Open Access Journals (Sweden)

    Jan Felix Drexler

    Full Text Available BACKGROUND: Henipaviruses (Hendra and Nipah virus are highly pathogenic members of the family Paramyxoviridae. Fruit-eating bats of the Pteropus genus have been suggested as their natural reservoir. Human Henipavirus infections have been reported in a region extending from Australia via Malaysia into Bangladesh, compatible with the geographic range of Pteropus. These bats do not occur in continental Africa, but a whole range of other fruit bats is encountered. One of the most abundant is Eidolon helvum, the African Straw-coloured fruit bat. METHODOLOGY/PRINCIPAL FINDINGS: Feces from E. helvum roosting in an urban setting in Kumasi/Ghana were tested for Henipavirus RNA. Sequences of three novel viruses in phylogenetic relationship to known Henipaviruses were detected. Virus RNA concentrations in feces were low. CONCLUSIONS/SIGNIFICANCE: The finding of novel putative Henipaviruses outside Australia and Asia contributes a significant extension of the region of potential endemicity of one of the most pathogenic virus genera known in humans.

  16. REDIdb: the RNA editing database.

    Science.gov (United States)

    Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

    2007-01-01

    The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.

  17. 5S rRNA-derived and tRNA-derived SINEs in fruit bats.

    Science.gov (United States)

    Gogolevsky, Konstantin P; Vassetzky, Nikita S; Kramerov, Dmitri A

    2009-05-01

    Most short retroposons (SINEs) descend from cellular tRNA of 7SL RNA. Here, four new SINEs were found in megabats (Megachiroptera) but neither in microbats nor in other mammals. Two of them, MEG-RS and MEG-RL, descend from another cellular RNA, 5S rRNA; one (MEG-T2) is a tRNA-derived SINE; and MEG-TR is a hybrid tRNA/5S rRNA SINE. Insertion locus analysis suggests that these SINEs were active in the recent fruit bat evolution. Analysis of MEG-RS and MEG-RL in comparison with other few 5S rRNA-derived SINEs demonstrates that the internal RNA polymerase III promoter is their most invariant region, while the secondary structure is more variable. The mechanisms underlying the modular structure of these and other SINEs as well as their variation are discussed. The scenario of evolution of MEG SINEs is proposed.

  18. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

    2017-01-01

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  19. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  20. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    Science.gov (United States)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  1. Role of RNase MRP in viral RNA degradation and RNA recombination.

    Science.gov (United States)

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  2. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

    Science.gov (United States)

    de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

    2016-01-01

    Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

  3. The use of 125iodine-labeled RNA for detection of the RNA binding to ribosomes

    International Nuclear Information System (INIS)

    Mori, Tomohiko; Fukuda, Mitsuru

    1975-01-01

    The in vitro labeling of RNA with radioactive iodine is the efficient method to obtain the RNA with high specific activity. The present paper reports on the application of this technique to the production of iodine-labeled RNA for use in the experiment of binding RNA to ribosomes. Tobacco mosaic virus (TMV) RNA was used as natural mRNA, and E. coli S-30 preparation was used as a source of ribosomes. The TMV-RNA was prepared by bentonite-phenol extraction from TMV, and the method used for the iodation of RNA was based on the procedure described by Getz et al. The iodine-labeled RNA was incubated in a cell-free protein synthesizing system (S-30) prepared from E. coli K-12. After the incubation, the reaction mixture was layered onto sucrose gradient, centrifuged, and fractionated into 18 fractions. Optical density at 260 nm was measured, and radioactivity was counted, for each fraction. The binding of mRNA to ribosomes occurred even at 0 deg C, and the occurrence of the nonspecific binding was also shown. Consequently, the specific binding, i.e. the formation of the initiation complex being involved in amino acid incorporation, may be estimated by subtracting the radioactivity associated with monosomes in the presence of both rRNA and ATA from that in the presence of rRNA only. It was shown that the iodine-labeled RNA can be used for the studies of binding RNA to ribosomes. (Kako, I.)

  4. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

    Science.gov (United States)

    Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

    2014-02-04

    TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

  5. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    Science.gov (United States)

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

  6. Argonaute: The executor of small RNA function.

    Science.gov (United States)

    Azlan, Azali; Dzaki, Najat; Azzam, Ghows

    2016-08-20

    The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  7. Designing synthetic RNA for delivery by nanoparticles

    International Nuclear Information System (INIS)

    Jedrzejczyk, Dominika; Pawlowska, Roza; Chworos, Arkadiusz; Gendaszewska-Darmach, Edyta

    2017-01-01

    The rapid development of synthetic biology and nanobiotechnology has led to the construction of various synthetic RNA nanoparticles of different functionalities and potential applications. As they occur naturally, nucleic acids are an attractive construction material for biocompatible nanoscaffold and nanomachine design. In this review, we provide an overview of the types of RNA and nucleic acid’s nanoparticle design, with the focus on relevant nanostructures utilized for gene-expression regulation in cellular models. Structural analysis and modeling is addressed along with the tools available for RNA structural prediction. The functionalization of RNA-based nanoparticles leading to prospective applications of such constructs in potential therapies is shown. The route from the nanoparticle design and modeling through synthesis and functionalization to cellular application is also described. For a better understanding of the fate of targeted RNA after delivery, an overview of RNA processing inside the cell is also provided. (topical review)

  8. Predicting RNA Structure Using Mutual Information

    DEFF Research Database (Denmark)

    Freyhult, E.; Moulton, V.; Gardner, P. P.

    2005-01-01

    , to display and predict conserved RNA secondary structure (including pseudoknots) from an alignment. Results: We show that MIfold can be used to predict simple pseudoknots, and that the performance can be adjusted to make it either more sensitive or more selective. We also demonstrate that the overall...... package. Conclusion: MIfold provides a useful supplementary tool to programs such as RNA Structure Logo, RNAalifold and COVE, and should be useful for automatically generating structural predictions for databases such as Rfam. Availability: MIfold is freely available from http......Background: With the ever-increasing number of sequenced RNAs and the establishment of new RNA databases, such as the Comparative RNA Web Site and Rfam, there is a growing need for accurately and automatically predicting RNA structures from multiple alignments. Since RNA secondary structure...

  9. Preparation of Total RNA from Fission Yeast.

    Science.gov (United States)

    Bähler, Jürg; Wise, Jo Ann

    2017-04-03

    Treatment with hot phenol breaks open fission yeast cells and begins to strip away bound proteins from RNA. Deproteinization is completed by multiple extractions with chloroform/isoamyl alcohol and separation of the aqueous and organic phases using MaXtract gel, an inert material that acts as a physical barrier between the phases. The final step is concentration of the RNA by ethanol precipitation. The protocol can be used to prepare RNA from several cultures grown in parallel, but it is important not to process too many samples at once because delays can be detrimental to RNA quality. A reasonable number of samples to process at once would be three to four for microarray or RNA sequencing analyses and six for preliminary investigations of mutants implicated in RNA metabolism. © 2017 Cold Spring Harbor Laboratory Press.

  10. A probabilistic model of RNA conformational space

    DEFF Research Database (Denmark)

    Frellsen, Jes; Moltke, Ida; Thiim, Martin

    2009-01-01

    efficient sampling of RNA conformations in continuous space, and with associated probabilities. We show that the model captures several key features of RNA structure, such as its rotameric nature and the distribution of the helix lengths. Furthermore, the model readily generates native-like 3-D......, the discrete nature of the fragments necessitates the use of carefully tuned, unphysical energy functions, and their non-probabilistic nature impairs unbiased sampling. We offer a solution to the sampling problem that removes these important limitations: a probabilistic model of RNA structure that allows......The increasing importance of non-coding RNA in biology and medicine has led to a growing interest in the problem of RNA 3-D structure prediction. As is the case for proteins, RNA 3-D structure prediction methods require two key ingredients: an accurate energy function and a conformational sampling...

  11. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

    Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

  12. The Old and New RNA World

    Directory of Open Access Journals (Sweden)

    Zofia Szweykowska-Kulińska

    2014-12-01

    Full Text Available Among the numerous hypotheses offering a scenario for the origin of life on Earth, the one called “The RNA World” has gained the most attention. According to this hypothesis RNA acted as a genetic information storage material, as a catalyst of all metabolic reactions, and as a regulator of all processes in the primordial world. Various experiments show that RNA molecules could have been synthesized abiotically, with the potential to mediate a whole repertoire of metabolic reactions. Ribozymes carrying out aminoacyl-tRNA reactions have been found in SELEX (systematic evolution of ligands by exponential enrichment approaches and the development of a ribosome from a RNA-built protoribosome is easy to imagine. Transfer RNA aminoacylation, protoribosome origin, and the availability of amino acids on early Earth allowed the genetic code to evolve. Encoded proteins most likely stabilized RNA molecules and were able to create channels across membranes. In the modern cell, DNA replaced RNA as the main depositor of genetic information and proteins carry out almost all metabolic reactions. However, RNA is still playing versatile, crucial roles in the cell. Apart from its classical functions in the cell, a huge small RNA world is controlling gene expression, chromatin condensation, response to environmental cues, and protecting the cell against the invasion of various nucleic acids forms. Long non-coding RNAs act as crucial gene expression regulators. Riboswitches act at the level of transcription, splicing or translation and mediate feedback regulation on biosynthesis and transport of the ligand they sense. Alternative splicing generates genetic variability and increases the protein repertoire in response to developmental or environmental changes. All these regulatory functions are essential in shaping cell plasticity in the changing milieu. Recent discoveries of new, unexpected and important functions of RNA molecules support the hypothesis that we

  13. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  14. Inverse folding of RNA pseudoknot structures

    Directory of Open Access Journals (Sweden)

    Li Linda YM

    2010-06-01

    Full Text Available Abstract Background RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and G-U-base pairings (secondary structure and additional cross-serial base pairs. These interactions are called pseudoknots and are observed across the whole spectrum of RNA functionalities. In the context of studying natural RNA structures, searching for new ribozymes and designing artificial RNA, it is of interest to find RNA sequences folding into a specific structure and to analyze their induced neutral networks. Since the established inverse folding algorithms, RNAinverse, RNA-SSD as well as INFO-RNA are limited to RNA secondary structures, we present in this paper the inverse folding algorithm Inv which can deal with 3-noncrossing, canonical pseudoknot structures. Results In this paper we present the inverse folding algorithm Inv. We give a detailed analysis of Inv, including pseudocodes. We show that Inv allows to design in particular 3-noncrossing nonplanar RNA pseudoknot 3-noncrossing RNA structures-a class which is difficult to construct via dynamic programming routines. Inv is freely available at http://www.combinatorics.cn/cbpc/inv.html. Conclusions The algorithm Inv extends inverse folding capabilities to RNA pseudoknot structures. In comparison with RNAinverse it uses new ideas, for instance by considering sets of competing structures. As a result, Inv is not only able to find novel sequences even for RNA secondary structures, it does so in the context of competing structures that potentially exhibit cross-serial interactions.

  15. Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

    Science.gov (United States)

    Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

    2010-11-01

    MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

  16. RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

    Science.gov (United States)

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-01-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  17. Peptides as catalysts in the RNA world

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Dörr, Mark; Luisi, Pier Luigi

    The emergence of RNA chains from prebiotic soup is considered a stumbling block in the RNA world theory (Orgel 2004). Both the activation of RNA monomers and their subsequent oligomerization is hard to achieve in accepted early Earth conditions, thus putting doubt on the prebiotic plausibility...... chemistry and the RNA world. Prebiotic soup likely contained complex mixtures of various molecules. Interaction of peptides and nucleotides shows that we should give more consideration to systems chemistry approach in the origin-of-life research. Gorlero M, Wieczorek R, Adamala K, Giorgi A, Schininà ME...

  18. Emerging connections between RNA and autophagy

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Lubas, Michal; Lund, Anders H

    2017-01-01

    in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge......Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority...... of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease....

  19. MiRNA Biogenesis and Intersecting Pathways

    DEFF Research Database (Denmark)

    Ben Chaabane, Samir

    MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Plant miRNAs are critical for plant growth, development and stress response, and are processed in Arabidopsis from primary miRNA transcripts (pri-miRNAs) by the endonuclease activity of the DICER-LIKE1...... questions need to be addressed to establish a valid link, we provide encouraging evidence of the involvement of chromatin remodeling factors FAS1 and FAS2 in miRNA biogenesis. Together, we have expanded our understanding of the intersections between miRNA biogenesis and other pathways....

  20. Personalized RNA Medicine for Pancreatic Cancer.

    Science.gov (United States)

    Gilles, Maud-Emmanuelle; Hao, Liangliang; Huang, Ling; Rupaimoole, Rajesha; Lopez-Casas, Pedro P; Pulver, Emilia; Jeong, Jong Cheol; Muthuswamy, Senthil K; Hidalgo, Manuel; Bhatia, Sangeeta N; Slack, Frank J

    2018-04-01

    Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors. Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars. Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734-47. ©2018 AACR . ©2018 American Association for Cancer Research.

  1. mRNA processing in yeast

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been pa