Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription
Janissen, Richard ; Arens, Mathia M.A. ; Vtyurina, Natalia N. ; Rivai, Zaïda ; Sunday, Nicholas D. ; Eslami-Mossallam, Behrouz ; Gritsenko, Alexey A. ; Laan, Liedewij ; Ridder, Dick de; Artsimovitch, Irina ; Dekker, Nynke H. ; Abbondanzieri, Elio A. ; Meyer, Anne S. - \ 2018
Cell 174 (2018)5. - ISSN 0092-8674 - p. 1188 - 1199.e14.
DNA condensation - Dps - magnetic tweezers - nucleoid - RNA polymerase - single-molecule biophysics - stationary phase - stress response - transcription

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.

Reporters for sensitive and quantitative measurement of auxin response
Liao, C.Y. ; Smet, W.M.S. ; Brunoud, G. ; Yoshida, S. ; Vernoux, T. ; Weijers, D. - \ 2015
Nature Methods : techniques for life scientists and chemists 12 (2015). - ISSN 1548-7091 - p. 207 - 210.
apical-basal axis - box protein tir1 - aux/iaa proteins - arabidopsis - transcription - expression - transport - specificity - sufficient - perception
The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.
BABY BOOM-induced somatic embryogenesis in Arabidopsis
Horstman, A. - \ 2015
University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462572317 - 233
arabidopsis - somatische embryogenese - in vitro kweek - cellen - weefselkweek - plantengroeiregulatoren - somatische embryo's - transcriptie - somatic embryogenesis - in vitro culture - cells - tissue culture - plant growth regulators - somatic embryos - transcription

Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes.

Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM.

BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited.

Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes.

A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE.

Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif.

Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes.

In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol.

Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways.

Reduced seed germination in Arabidopsis over-expressing SWI/SNF2 ATPase genes
Leeggangers, H.A.C.F. ; Folta, A. ; Muras, A. ; Nap, J.P.H. ; Mlynarova, L. - \ 2015
Physiologia Plantarum 153 (2015)2. - ISSN 0031-9317 - p. 318 - 326.
thaliana - dormancy - transcription - maturation - stress - arrest - growth - rna
In the life of flowering plants, seed germination is a critical step to ensure survival into the next generation. Generally the seed prior to germination has been in a dormant state with a low rate of metabolism. In the transition from a dormant seed to a germinating seed, various epigenetic mechanisms play a regulatory role. Here, we demonstrate that the over-expression of chromatin remodeling ATPase genes (AtCHR12 or AtCHR23) reduced the frequency of seed germination in Arabidopsis thaliana up to 30% relative to the wild-type seeds. On the other hand, single loss-of-function mutations of the two genes did not affect seed germination. The reduction of germination in over-expressing mutants was more pronounced in stress conditions (salt or high temperature), showing the impact of the environment. Reduced germinations upon over-expression coincided with increased transcript levels of seed maturation genes and with reduced degradation of their mRNAs stored in dry seeds. Our results indicate that repression of AtCHR12/23 gene expression in germinating wild-type Arabidopsis seeds is required for full germination. This establishes a functional link between chromatin modifiers and regulatory networks towards seed maturation and germination.
Paramyxovirus-based producton of Rift Valley fever virus replicon particles
Wichgers Schreur, P.J. ; Oreshkova, N. ; Harders, F. ; Bossers, A. ; Moormann, R.J.M. ; Kortekaas, J.A. - \ 2014
Journal of General Virology 95 (2014)12. - ISSN 0022-1317 - p. 2638 - 2648.
newcastle-disease virus - hemagglutinin-neuraminidase protein - fusion protein - vaccine vector - nsm protein - hn protein - virulence - transcription - bunyaviridae - expression
Replicon-particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR), and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) were able to produce high levels of NSR particles. In addition, using reverse genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains two amino acid substitutions in, respectively, Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.
Probing the relation between protein–protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS
Giangrossi, M. ; Wintraecken, K. ; Spurio, R. ; Vries, R.J. de - \ 2014
Biochimica et Biophysica Acta. Proteins & Proteomics 1844 (2014)2. - ISSN 1570-9639 - p. 339 - 345.
in-vivo oligomerization - virulence gene icsa - escherichia-coli - curved dna - structuring protein - shigella-flexneri - organization - domain - mechanism - transcription
We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein–protein interactions. These results emphasize the relative contributions of protein–protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.
Cell proliferation and modulation of interaction of estrogen receptors with coregulators induced by ERa and ERB agonists
Evers, N.M. ; Berg, J.H.J. van den; Wang, S. ; Melchers, D. ; Houtman, J. ; Haan, L.H.J. de; Ederveen, A.G.H. ; Groten, J.P. ; Rietjens, I. - \ 2014
Journal of Steroid Biochemistry and Molecular Biology 143 (2014). - ISSN 0960-0760 - p. 376 - 385.
breast-cancer-cells - expression - coactivator - mechanisms - ligands - genes - phytoestrogens - transcription - antagonist - (er)alpha
The aim of the present study was to investigate modulation of the interaction of the ERa and ERß with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERa and ERß agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERa and ERß mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERß cells with variable ERa/ERß ratio, and finally for ligand dependent modulation of the interaction of ERa and ERß with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERa/ERß ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERa agonists activate cell proliferation whereas ERß suppresses ERa mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERa or ERß activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERa or ERß activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays. It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERa and ERß and the cellular ERa/ERß ratio whereas differences in the modulation of the interaction of the ERa and ERß with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.
Induction of indirect plant defense in the context of multiple herbivory : gene transcription, volatile emission, and predator behavior
Menzel, T.R. - \ 2014
University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462571297 - 146
planten - plaagresistentie - geïnduceerde resistentie - verdedigingsmechanismen - multitrofe interacties - phaseolus lunatus - mijten - tetranychus urticae - roofmijten - phytoseiulus persimilis - voedingsgedrag - genen - transcriptie - genexpressie - herbivoor-geinduceerde plantengeuren - plants - pest resistance - induced resistance - defence mechanisms - multitrophic interactions - mites - predatory mites - feeding behaviour - genes - transcription - gene expression - herbivore induced plant volatiles

Abstract

Plants live in complex environments and are under constant threat of being attacked by herbivorous arthropods. Consequently plants possess an arsenal of sophisticated mechanisms in order to defend themselves against their ubiquitous attackers. Induced indirect defenses involve the attraction of natural enemies of herbivores, such as predators and parasitoids. Predators and parasitoids use odors emitted by damaged plants that serve as a “cry for help” to find their respective prey or host herbivore. The aim of this thesis was to use a multidisciplinary approach, with focus on molecular and chemical methods, combined with behavioral investigations, to elucidate the mechanisms of plant responses to multiple herbivory that affect a tritrophic system consisting of a plant, an herbivore and a natural enemy.

Induced plant defenses are regulated by a network of defense signaling pathways in which phytohormones act as signaling molecules. Accordingly, simulation of herbivory by exogenous application of phytohormones and actual herbivory by the two-spotted spider mite Tetranychus urticae affected transcript levels of a defense gene involved in indirect defense in Lima bean. However, two other genes involved in defense were not affected at the time point investigated. Moreover, application of a low dose of JA followed by minor herbivory by T. urticae spider mites affected gene transcript levels and emissions of plant volatiles commonly associated with herbivory. Only endogenous phytohormone levels of jasmonic acid (JA), but not salicylic acid (SA), were affected by treatments. Nevertheless, the low-dose JA application resulted in a synergistic effect on gene transcription and an increased emission of a volatile compound involved in indirect defense after herbivore infestation.

Caterpillar feeding as well as application of caterpillar oral secretion on mechanically inflicted wounds are frequently used to induce plant defense against biting-chewing insects, which is JA-related. Feeding damage by two caterpillar species caused mostly identical induction of gene transcription, but combination of mechanical damage and oral secretions of caterpillars caused differential induction of the transcription of defense genes. Nevertheless, gene transcript levels for plants that subsequently experienced an infestation by T. urticae were only different for a gene potentially involved in direct defense of plants that experienced a single event of herbivory by T. urticae. Indirect defense was not affected. Also sequential induction of plant defense by caterpillar oral secretion and an infestation by T. urticae spider mites did not interfere with attraction of the specialist predatory mite P. persimilis in olfactometer assays. The predator did distinguish between plants induced by spider mites and plants induced by the combination of mechanical damage and caterpillar oral secretion but not between plants with single spider mite infestation and plants induced by caterpillar oral secretion prior to spider mite infestation. The composition of the volatile blends emitted by plants induced by spider mites only or by the sequential induction treatment of caterpillar oral secretion followed by spider mite infestation were similar. Consequently, the induction of plant indirect defense as applied in these experiments was not affected by previous treatment with oral secretion of caterpillars. Moreover, herbivory by conspecific T. urticae mites did not affect gene transcript levels or emission of volatiles of plants that experienced two bouts of herbivore attack by conspecific spider mites compared to plants that experienced only one bout of spider mite attack. This suggests that Lima bean plants do no increase defense in response to sequential herbivory by T. urticae.

In conclusion, using a multidisciplinary approach new insights were obtained in the mechanisms of induction of indirect plant defense and tritrophic interactions in a multiple herbivore context, providing helpful leads for future research on plant responses to multiple stresses.

Comparative efficacy of two next-generation Rift Valley fever vaccines
Kortekaas, J.A. ; Oreshkova, N. ; Keulen, L.J.M. van; Kant, J. ; Bosch, B.J. ; Bouloy, M. ; Moulin, V. ; Goovaerts, D. ; Moormann, R.J.M. - \ 2014
Vaccine 32 (2014). - ISSN 0264-410X - p. 4901 - 4908.
nss protein - down-regulation - mp-12 vaccine - virus-vaccine - safety - transcription - sheep - immunogenicity - attenuation - rna
Rift Valley fever virus (RVFV) is a re-emerging zoonotic bunyavirus of the genus Phlebovirus. A natural isolate containing a large attenuating deletion in the small (S) genome segment previously yielded a highly effective vaccine virus, named Clone 13. The deletion in the S segment abrogates expression of the NSs protein, which is the major virulence factor of the virus. To develop a vaccine of even higher safety, a virus named R566 was created by natural laboratory reassortment. The R566 virus combines the S segment of the Clone 13 virus with additional attenuating mutations on the other two genome segments M and L, derived from the previously created MP-12 vaccine virus. To achieve the same objective, a nonspreading RVFV (NSR-Gn) was created by reverse-genetics, which not only lacks the NSs gene but also the complete M genome segment. We have now compared the vaccine efficacies of these two next-generation vaccines and included the Clone 13 vaccine as a control for optimal efficacy. Groups of eight lambs were vaccinated once and challenged three weeks later. All mock-vaccinated lambs developed high fever and viremia and three lambs did not survive the infection. As expected, lambs vaccinated with Clone 13 were protected from viremia and clinical signs. Two lambs vaccinated with R566 developed mild fever after challenge infection, which was associated with low levels of viral RNA in the blood, whereas vaccination with the NSR-Gn vaccine completely prevented viremia and clinical signs.
Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato
Yang, X. ; Caro Rios, C.M. ; Hutton, S.F. ; Scott, J.W. ; Guo, Y. ; Wang, Xiaoxuan ; Rashid, H. ; Szinay, D. ; Jong, J.H.S.G.M. de; Visser, R.G.F. ; Bai, Y. ; Du, Y. - \ 2014
Molecular Breeding 34 (2014)2. - ISSN 1380-3743 - p. 749 - 760.
cultivated tomato - rna - introgression - recombination - transcription - replication - encodes - locus
Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanumlycopersicum) fromwild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession ‘‘B6013,’’ that confers resistance to TYLCV was previously mapped to a 19-cMregion on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flankingmarkersC2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Severalmarkers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.
Transcriptional regulation of nodule development and senescence in Medicago truncatula
Karmarkar, V.M. - \ 2014
University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789462570214 - 110
medicago truncatula - plantenontwikkeling - veroudering - wortelknolletjes - stikstoffixatie - genexpressie - symbiose - transcriptie - transcriptiefactoren - plant development - senescence - root nodules - nitrogen fixation - gene expression - symbiosis - transcription - transcription factors
Differential translation tunes uneven production of operon-encoded proteins
Quax, T.E.F. ; Wolf, Y.I. ; Koehorst, J.J. ; Wurtzel, O. ; Oost, R. van der; Ran, W. ; Blombach, F. ; Makarova, K.S. ; Brouns, S.J.J. ; Forster, A.C. ; Wagner, E.G.H. ; Sorek, R. ; Koonin, E.V. ; Oost, J. van der - \ 2013
Cell Reports 4 (2013)5. - ISSN 2211-1247 - p. 938 - 944.
escherichia-coli - gene-expression - messenger-rnas - codon bias - transcription - sequence - ribosome - bacteria - genome - architecture
Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus
Staals, R.H.J. ; Agari, Y. ; Maki-Yonekura, S. ; Zhu, Y. ; Taylor, D.W. ; Duijn, E. van; Barendregt, A. ; Vlot, M. ; Koehorst, J.J. ; Sakamoto, K. ; Masuda, A. ; Dohmae, N. ; Schaap, P.J. ; Doudna, J.A. ; Heck, A. ; Yonekura, K. ; Oost, J. van der; Shinkai, A. - \ 2013
Molecular Cell 52 (2013)1. - ISSN 1097-2765 - p. 135 - 145.
of-flight instrument - mass-spectrometry - escherichia-coli - silencing complex - antiviral defense - immune-system - protein - interference - transcription - recognition
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes
Genome-wide gene expression analysis of anguillid herpesvirus 1
Beurden, S.J. van; Peeters, B.P.H. ; Rottier, P.J.M. ; Davison, A.A. ; Engelsma, M.Y. - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164 - 11 p.
channel catfish virus - time rt-pcr - dna microarray - european eel - murine gammaherpesvirus-68 - transcription - persistence - proteins - carp
Background Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR. Results Four immediate-early genes – open reading frames 1, 6A, 127 and 131 – were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins. Conclusions Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.
The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling
Fros, J.J. ; Maten, E. van der; Vlak, J.M. ; Pijlman, G.P. - \ 2013
Journal of Virology 87 (2013)18. - ISSN 0022-538X - p. 10394 - 10400.
semliki-forest-virus - nonstructural protein-2 - nuclear-localization - infection - transcription - alphaviruses - translation - expression - mutations - cells
Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling
Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function
Outchkourov, N.S. ; Muino Acuna, J.M. ; Kaufmann, K. ; IJken, W.F.J. ; Groot Koerkamp, M.J. ; Leenen, D. van; Graaf, P. de; Holstege, F.C.P. ; Grosveld, F. ; Timmers, H.T.M. - \ 2013
Cell Reports 3 (2013)4. - ISSN 2211-1247 - p. 1071 - 1079.
little-imaginal-discs - embryonic stem-cells - binding-protein 2 - gene-expression - distinct functions - self-renewal - human genome - transcription - differentiation - reveals
The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters
MADS interactomics : towards understanding the molecular mechanisms of plant MADS-domain transcription factor function
Smaczniak, C.D. - \ 2013
University. Promotor(en): Gerco Angenent, co-promotor(en): Kerstin Kaufmann. - S.l. : s.n. - ISBN 9789461734525 - 178
planten - moleculaire biologie - transcriptie - transcriptiefactoren - arabidopsis - mads-box eiwitten - regulatoren - dna-bindende eiwitten - plants - molecular biology - transcription - transcription factors - mads-box proteins - regulators - dna binding proteins

Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of transcription factors present in all eukaryotes. In plants, and especially in seed plants, this family has significantly expanded. For example, more than 100 representatives are found in the Arabidopsis genome. MADS-box genes have initially been shown to play major roles in flower development, however their emerging functional characterization revealed functions in almost all developmental processes throughout the plant life cycle. How MADS-domain transcription factors acquire their functional specificity remains unresolved. The goal of this thesis was to characterize some of the molecular mechanisms by which MADS-domain proteins act in Arabidopsis.

Chapter 1 comprehensively reviews functions of MADS-domain transcription factors in flowering plants, with a main focus on Arabidopsis. Major classes of MADS-domain proteins are introduced, and their modular structures are described. Additionally, it is shown that several distinctive subfamilies of MADS-box genes can be inferred from the phylogenetic analysis of the whole gene family. By compiling recent studies on MADS-domain protein-protein and protein-DNA interactions, we present a hypothetical model of MADS-domain protein action that combines higher-order protein complex formation and active chromatin remodeling by large transcriptional machineries.

Chapter 2 describes MADS-domain protein complexes that are potentially formed during Arabidopsis flower development. By using a targeted proteomics approach we were able to characterize the protein interactome of major floral homeotic MADS-domain proteins (APETALA1, APETALA3, PISTILLATA, AGAMOUS, SEPALLATA3 and FRUITFULL) in native plant tissues, confirming interactions suggested in the ‘floral quartet’ model. Additionally, we discovered transcription factors from other families and chromatin-associated proteins as possible interaction partners of MADS-domain proteins. These interactions shed light on the combinatorial modes of action of MADS-domain transcription factors and suggest that they can act by recruiting or redirecting the chromatin remodeling machinery to control the expression of their target genes.

In Chapter 3 we review recent advances in proteomics approaches used to study cellular signaling and developmental processes in plants. We mention the emerging tools for of whole plant proteome characterization as well as sub-cellular protein localization. The major focus, though, is on the description of complete cellular signaling cascades in plants, starting from the characterization of signaling mobile molecules (e.g. peptide or protein), through identification of receptors and receptor protein complexes, ending with identification of intermediate signaling pathway members. Two examples of biochemical procedures used to identify complexes of membrane-bound receptors and transcriptional regulators from nuclei are described in Chapter 4. In our optimized method we make use of fluorophore-tagged single step affinity purification of protein complexes and label-free mass spectrometry-based protein quantification to distinguish true complex partners from non-specifically precipitated proteins.

The exact molecular mechanisms of DNA sequence recognition by MADS-domain transcription factors are still unknown. Particularly intriguing is the question whether various MADS-domain protein complexes possess different DNA-binding specificities. We address this question in Chapter 5. We used systematic evolution of ligands by exponential enrichment (SELEX) followed by high-throughput sequencing (seq) approach to discriminate DNA-binding specificities of several MADS-domain protein homo- and heterodimers.

Finally, in Chapter 6, we aimed to identify the molecular features of different DNA-binding specificities of MADS-domain transcription factors.With help of bioinformatics tools and in vitro DNA-binding assays we found that structural characteristics of the DNA play an important role in DNA-binding of MADS-domain proteins.

Taken together, research described in this thesis advances our knowledge on the molecular mechanisms of MADS-domain transcription factor action in plants. Chapter 7 concludes the thesis and describes future perspectives in MADS-domain protein research. Highlighted are the advances of high-throughput (proteomics and genomics) technologies that could be used to unravel not only the static characteristics of transcriptional regulation but also the dynamic and stoichiometric changes of complex protein and gene regulatory networks during plant development.

Mycorrhizal symbiosis: ancient signalling mechanisms co-opted
Geurts, R. ; Vleeshouwers, V.G.A.A. - \ 2012
Current Biology 22 (2012)23. - ISSN 0960-9822 - p. R997 - R999.
medicago-truncatula - transcription - requires - family - cutin - nsp1
Mycorrhizal root endosymbiosis is an ancient property of land plants. Two parallel studies now provide novel insight into the mechanism driving this interaction and how it is used by other filamentous microbes like pathogenic oomycetes.
A baculovirus photolyase with DNA repair activity and circadian clock regulatory function
Biernat, M.A. ; Eker, A.P.M. ; Oers, M.M. van; Vlak, J.M. ; Horst, G.T.J. van der; Chaves, I. - \ 2012
Journal of Biological Rhythms 27 (2012)1. - ISSN 0748-7304 - p. 3 - 11.
chrysodeixis-chalcites nucleopolyhedrovirus - cryptochrome/photolyase family - mammalian cry1 - evolution - animals - plants - magnetoreception - identification - transcription - oscillator
Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses.
Efficacy of three candidate Rift Valley fever vaccines in sheep
Kortekaas, J.A. ; Antonis, A.F.G. ; Kant-Eenbergen, H.C.M. ; Vloet, R.P.M. ; Vogel-Brink, A. ; Oreshkova, N.D. ; Boer, S.M. de; Bosch, B.J. ; Moormann, R.J.M. - \ 2012
Vaccine 30 (2012)23. - ISSN 0264-410X - p. 3423 - 3429.
north-american mosquitos - enzootic hepatitis - rhesus macaques - virus - disease - protein - transcription - protection - challenge - africa
Rift Valley fever virus (RVFV) is a mosquito-transmitted Bunyavirus that causes high morbidity and mortality among ruminants and humans. The virus is endemic to the African continent and the Arabian Peninsula and continues to spread into new areas. The explosive nature of RVF outbreaks requires that vaccines provide swift protection after a single vaccination. We recently developed several candidate vaccines and here report their efficacy in lambs within three weeks after a single vaccination. The first vaccine comprises the purified ectodomain of the Gn structural glycoprotein formulated in a water-in-oil adjuvant. The second vaccine is based on a Newcastle disease virus-based vector that produces both RVFV structural glycoproteins Gn and Gc. The third vaccine comprises a recently developed nonspreading RVFV. The latter two vaccines were administered without adjuvant. The inactivated whole virus-based vaccine produced by Onderstepoort Biological Products was used as a positive control. Five out of six mock-vaccinated lambs developed high viremia and fever and one lamb succumbed to the challenge infection. A single vaccination with each vaccine resulted in a neutralizing antibody response within three weeks after vaccination and protected lambs from viremia, pyrexia and mortality.
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