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  • 1.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Gharizadeh, B.
    O'Meara, D.
    Odeberg, Jacob
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Genotyping by apyrase-mediated allele-specific extension2001In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 24Article in journal (Refereed)
    Abstract [en]

    This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

  • 2.
    Alexeyenko, Andrey
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schmitt, Thomas
    Tjärnberg, Andreas
    Stockholm University, Science for Life Laboratory.
    Guala, Dmitri
    Stockholm University, Science for Life Laboratory.
    Frings, Oliver
    Stockholm University, Science for Life Laboratory.
    Sonnhammer, Erik L. L.
    Stockholm University, Science for Life Laboratory.
    Comparative interactomics with Funcoup 2.02012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no D1, p. D821-D828Article in journal (Refereed)
    Abstract [en]

    FunCoup (http://FunCoup.sbc.su.se) is a database that maintains and visualizes global gene/protein networks of functional coupling that have been constructed by Bayesian integration of diverse high-throughput data. FunCoup achieves high coverage by orthology-based integration of data sources from different model organisms and from different platforms. We here present release 2.0 in which the data sources have been updated and the methodology has been refined. It contains a new data type Genetic Interaction, and three new species: chicken, dog and zebra fish. As FunCoup extensively transfers functional coupling information between species, the new input datasets have considerably improved both coverage and quality of the networks. The number of high-confidence network links has increased dramatically. For instance, the human network has more than eight times as many links above confidence 0.5 as the previous release. FunCoup provides facilities for analysing the conservation of subnetworks in multiple species. We here explain how to do comparative interactomics on the FunCoup website.

  • 3. Alkema, W. B. L.
    et al.
    Johansson, O.
    Lagergren, Jens
    KTH, Superseded Departments (pre-2005), Numerical Analysis and Computer Science, NADA.
    Wasserman, W. W.
    MSCAN: identification of functional clusters of transcription factor binding sites2004In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 32, p. W195-W198Article in journal (Refereed)
    Abstract [en]

    Identification of functional transcription factor binding sites in genomic sequences is notoriously difficult. The critical problem is the low specificity of predictions, which directly reflects the low target specificity of DNA binding proteins. To overcome the noise produced in predictions of individual binding sites, a new generation of algorithms achieves better predictive specificity by focusing on locally dense clusters of binding sites. MSCAN is a leading method for binding site cluster detection that determines the significance of observed sites while correcting for local compositional bias of sequences. The algorithm is highly flexible, applying any set of input binding models to the analysis of a user-specified sequence. From the user's perspective, a key feature of the system is that no reference data sets of regulatory sequences from co-regulated genes are required to train the algorithm. The output from MSCAN consists of an ordered list of sequence segments that contain potential regulatory modules. We have chosen the features in MSCAN such that sequence and matrix retrieval is highly facilitated, resulting in a web server that is intuitive to use. MSCAN is available at http://mscan.cgb.ki.se/cgi-bin/MSCAN.

  • 4. Anders, Gerd
    et al.
    Mackowiak, Sebastian D
    Jens, Marvin
    Maaskola, Jonas
    Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology, Max Delbrück Centre for Molecular Medicine, 13125 Berlin, Germany.
    Kuntzagk, Andreas
    Rajewsky, Nikolaus
    Landthaler, Markus
    Dieterich, Christoph
    doRiNA: a database of RNA interactions in post-transcriptional regulation.2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no Database issueArticle in journal (Refereed)
    Abstract [en]

    In animals, RNA binding proteins (RBPs) and microRNAs (miRNAs) post-transcriptionally regulate the expression of virtually all genes by binding to RNA. Recent advances in experimental and computational methods facilitate transcriptome-wide mapping of these interactions. It is thought that the combinatorial action of RBPs and miRNAs on target mRNAs form a post-transcriptional regulatory code. We provide a database that supports the quest for deciphering this regulatory code. Within doRiNA, we are systematically curating, storing and integrating binding site data for RBPs and miRNAs. Users are free to take a target (mRNA) or regulator (RBP and/or miRNA) centric view on the data. We have implemented a database framework with short query response times for complex searches (e.g. asking for all targets of a particular combination of regulators). All search results can be browsed, inspected and analyzed in conjunction with a huge selection of other genome-wide data, because our database is directly linked to a local copy of the UCSC genome browser. At the time of writing, doRiNA encompasses RBP data for the human, mouse and worm genomes. For computational miRNA target site predictions, we provide an update of PicTar predictions.

  • 5.
    Arif, Muhammad
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. School of Pharmaceutical Sciences & Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, Zhengzhou University, Zhengzhou, Henan Province, PR 450001, China.
    Li, Xiangyu
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Güngör, Cem
    Çakmak, Buğra
    Arslantürk, Metin
    Tebani, Abdellah
    Özcan, Berkay
    Subaş, Oğuzhan
    Zhou, Wenyu
    Piening, Brian
    Turkez, Hasan
    Fagerberg, Linn
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Price, Nathan
    Hood, Leroy
    Snyder, Michael
    Nielsen, Jens
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. Centre for Host–Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King's College London, London SE1 9RT, UK.
    iNetModels 2.0: an interactive visualization and database of multi-omics data.2021In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no W1, p. W271-W276, article id gkab254Article in journal (Refereed)
    Abstract [en]

    It is essential to reveal the associations between various omics data for a comprehensive understanding of the altered biological process in human wellness and disease. To date, very few studies have focused on collecting and exhibiting multi-omics associations in a single database. Here, we present iNetModels, an interactive database and visualization platform of Multi-Omics Biological Networks (MOBNs). This platform describes the associations between the clinical chemistry, anthropometric parameters, plasma proteomics, plasma metabolomics, as well as metagenomics for oral and gut microbiome obtained from the same individuals. Moreover, iNetModels includes tissue- and cancer-specific Gene Co-expression Networks (GCNs) for exploring the connections between the specific genes. This platform allows the user to interactively explore a single feature's association with other omics data and customize its particular context (e.g. male/female specific). The users can also register their data for sharing and visualization of the MOBNs and GCNs. Moreover, iNetModels allows users who do not have a bioinformatics background to facilitate human wellness and disease research. iNetModels can be accessed freely at https://inetmodels.com without any limitation.

  • 6.
    Azuara, Cyril
    et al.
    Institut Pasteur, Paris France.
    Lindahl, Erik
    Stockholm University.
    Koehl, Patrice
    University of California, Davis.
    Orland, Henri
    Institut Pasteur, Paris, France.
    Delarue, Marc
    Institut Pasteur, Paris, France.
    PDB_Hydro: incorporating dipolar solvents with variable density in the Poisson-Boltzmann treatment of macromolecule electrostatics.2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no Web Server issue, p. W38-42Article in journal (Refereed)
    Abstract [en]

    We describe a new way to calculate the electrostatic properties of macromolecules which eliminates the assumption of a constant dielectric value in the solvent region, resulting in a Generalized Poisson-Boltzmann-Langevin equation (GPBLE). We have implemented a web server (http://lorentz.immstr.pasteur.fr/pdb_hydro.php) that both numerically solves this equation and uses the resulting water density profiles to place water molecules at preferred sites of hydration. Surface atoms with high or low hydration preference can be easily displayed using a simple PyMol script, allowing for the tentative prediction of the dimerization interface in homodimeric proteins, or lipid binding regions in membrane proteins. The web site includes options that permit mutations in the sequence as well as reconstruction of missing side chain and/or main chain atoms. These tools are accessible independently from the electrostatics calculation, and can be used for other modeling purposes. We expect this web server to be useful to structural biologists, as the knowledge of solvent density should prove useful to get better fits at low resolution for X-ray diffraction data and to computational biologists, for whom these profiles could improve the calculation of interaction energies in water between ligands and receptors in docking simulations.

  • 7.
    Bandmann, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Combinatorial expression vector engineering for tuning of recombinant protein production in Escherichi coli2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5Article in journal (Refereed)
    Abstract [en]

    The complex and integrated nature of both genetic and protein level factors influencing recombinant protein production in Escherichia coli makes it difficult to predict the optimal expression strategy for a given protein. Here, two combinatorial library strategies were evaluated for their capability of tuning recombinant protein production in the cytoplasm of E. coli. Large expression vector libraries were constructed through either conservative (ExLib1) or free (ExLib2) randomization of a seven-amino-acid window strategically located between a degenerated start codon and a sequence encoding a fluorescently tagged target protein. Flow cytometric sorting and analyses of libraries, subpopulations or individual clones were followed by SDS-PAGE, western blotting, mass spectrometry and DNA sequencing analyses. For ExLib1, intracellular accumulation of soluble protein was shown to be affected by codon specific effects at some positions of the common N-terminal extension. Interestingly, for ExLib2 where the same sequence window was randomized via seven consecutive NN(G/T) tri-nucleotide repeats, high product levels (up to 24-fold higher than a reference clone) were associated with a preferential appearance of novel SID-like sequences. Possible mechanisms behind the observed effects are discussed.

  • 8.
    Behle, Anna
    et al.
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1; Photanol B.V, Science Park 406, 1098 XH Amsterdam, The Netherlands, Science Park 406.
    Dietsch, Maximilian
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Goldschmidt, Louis
    Institut f. Quantitative u. Theoretische Biologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Murugathas, Wandana
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Berwanger, Lutz C.
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Burmester, Jonas
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Yao, Lun
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Brandt, David
    Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstrasse 27, 33615 Bielefeld, Germany, Universitätsstrasse 27.
    Busche, Tobias
    Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstrasse 27, 33615 Bielefeld, Germany, Universitätsstrasse 27.
    Kalinowski, Jörn
    Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstrasse 27, 33615 Bielefeld, Germany, Universitätsstrasse 27.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Ebenhöh, Oliver
    Institut f. Quantitative u. Theoretische Biologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1; Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany, Universitätsstraße 1.
    Axmann, Ilka M.
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Machne, Rainer
    Institut f. Synthetische Mikrobiologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1; Institut f. Quantitative u. Theoretische Biologie, Heinrich-Heine Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany, Universitätsstrasse 1.
    Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 22, p. 12790-12808Article in journal (Refereed)
    Abstract [en]

    In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

  • 9. Bjorklund, Marcus Gry
    et al.
    Natanaelsson, Christian
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hao, Yong
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Microarray analysis using disiloxyl 70mer oligonucleotides2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 4, p. 1334-1342Article in journal (Refereed)
    Abstract [en]

    DNA microarray technology has evolved dramatically in recent years, and is now a common tool in researchers portfolios. The scope of the technique has expanded from small-scale studies to extensive studies such as classification of disease states. Technical knowledge regarding solid phase microarrays has also increased, and the results acquired today are more reliable than those obtained just a few years ago. Nevertheless, there are various aspects of microarray analysis that could be improved. In this article we show that the proportions of full-length probes used significantly affects the results of global analyses of transcriptomes. In particular, measurements of transcripts in low abundance are more sensitive to truncated probes, which generally increase the degree of cross hybridization and loss of specific signals. In order to improve microarray analysis, we here introduce a disiloxyl purification step, which ensures that all the probes on the microarray are at full length. We demonstrate that when the features on microarrays consist of full-length probes the signal intensity is significantly increased. The overall increase in intensity enables the hybridization stringency to be increased, and thus enhance the robustness of the results.

  • 10. Cristovao, Michele
    et al.
    Sisamakis, Evangelos
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hingorani, Manju M.
    Marx, Andreas D.
    Jung, Caroline P.
    Rothwell, Paul J.
    Seidel, Claus A. M.
    Friedhoff, Peter
    Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 12, p. 5448-5464Article in journal (Refereed)
    Abstract [en]

    Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.

  • 11. Dierckx, Anke
    et al.
    Dinér, Peter
    Department of Chemistry, Medicinal Chemistry, University of Gothenburg.
    El-Sagheer, A. H.
    Kumar, Joshi Dhruval
    Brown, T.
    Grøtli, Morten
    Wilhelmsson, Marcus
    Wilhelmsson, L Marcus
    Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 10, p. 4513-4524Article in journal (Refereed)
  • 12. Eisenhut, P.
    et al.
    Mebrahtu, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Moradi Barzadd, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Thalén, Niklas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Klanert, G.
    Weinguny, M.
    Sandegren, A.
    Su, C.
    Hatton, D.
    Borth, N.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories2020In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 20, article id e119Article in journal (Refereed)
    Abstract [en]

    Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

  • 13.
    Fouquier d'Hérouel, Aymeric
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Wessner, Françoise
    Halpern, David
    Ly-Vu, Joseph
    Kennedy, Sean P
    Serror, Pascale
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Repoila, Francis
    A simple and efficient method to search for selected primary transcripts: non-coding and antisense RNAs in the human pathogen Enterococcus faecalis2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 7, p. E46-Article in journal (Refereed)
    Abstract [en]

    Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5'RACE-derivative method coined '5'tagRACE', to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5'tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5'tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin-antitoxin modules.

  • 14.
    Gharizadeh, Baback
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Käller, Max
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Andersson,, Anders F.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Ahmadian, Afshin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 22, p. e146-Article in journal (Refereed)
    Abstract [en]

     Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

  • 15. Himanen, S. V.
    et al.
    Puustinen, M. C.
    Da Silva, A. J.
    Vihervaara, Anniina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sistonen, L.
    HSFs drive transcription of distinct genes and enhancers during oxidative stress and heat shock2022In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 50, no 11, p. 6102-6115Article in journal (Refereed)
    Abstract [en]

    Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 localizes to a unique set of promoters and enhancers to trans-activate oxidative stress-specific genes. Taken together, we show that HSFs function as multi-stress-responsive factors that activate distinct genes and enhancers when encountering changes in temperature and redox state. 

  • 16.
    Hu, Francis Jingxin
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lundqvist, Magnus
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Tech Univ Denmark, Novo Nord Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping2019In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 6, article id e34Article in journal (Refereed)
    Abstract [en]

    Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.

  • 17.
    Hultin, Emelie
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Käller, Max
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Competitive enzymatic reaction to control allele-specific extensions2005In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 5, p. e48:1-e48:10Article in journal (Refereed)
    Abstract [en]

    Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian, A., Gharizadeh, B., O'Meara, D., Odeberg, J. and Lundeberg, J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 50 end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

  • 18.
    Höjer, Pontus
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frick, Tobias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Siga, Humam
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Pourbozorgi, Parham
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Martin, Marcel
    Stockholm University, Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, SE-171 65, Solna, Sweden.
    Ahmadian, Afshin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    BLR: a flexible pipeline for haplotype analysis of multiple linked-read technologies2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 22, p. 114-114Article in journal (Refereed)
    Abstract [en]

    Linked-read sequencing promises a one-method approach for genome-wide insights including single nucleotide variants (SNVs), structural variants, and haplotyping. We introduce Barcode Linked Reads (BLR), an open-source haplotyping pipeline capable of handling millions of barcodes and data from multiple linked-read technologies including DBS, 10× Genomics, TELL-seq and stLFR. Running BLR on DBS linked-reads yielded megabase-scale phasing with low (<0.2%) switch error rates. Of 13616 protein-coding genes phased in the GIAB benchmark set (v4.2.1), 98.6% matched the BLR phasing. In addition, large structural variants showed concordance with HPRC-HG002 reference assembly calls. Compared to diploid assembly with PacBio HiFi reads, BLR phasing was more continuous when considering switch errors. We further show that integrating long reads at low coverage (∼10×) can improve phasing contiguity and reduce switch errors in tandem repeats. When compared to Long Ranger on 10× Genomics data, BLR showed an increase in phase block N50 with low switch-error rates. For TELL-Seq and stLFR linked reads, BLR generated longer or similar phase block lengths and low switch error rates compared to results presented in the original publications. In conclusion, BLR provides a flexible workflow for comprehensive haplotype analysis of linked reads from multiple platforms.

  • 19. Ivanov, Maxim
    et al.
    Kals, Mart
    Lauschke, Volker
    Barragan, Isabel
    Ewels, Philip
    Kaller, Max
    KTH, School of Biotechnology (BIO), Gene Technology.
    Axelsson, Tomas
    Lehtio, Janne
    Milani, Lili
    Ingelman-Sundberg, Magnus
    Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 14, p. 6756-6769Article in journal (Refereed)
    Abstract [en]

    To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene-and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.

  • 20.
    Kitamura, Akira
    et al.
    Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University , Sapporo , Japan.
    Tornmalm, Johan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Demirbay, Baris
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Piguet, Joachim
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Kinjo, Masataka
    Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University , Sapporo , Japan.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogeneous RNA G-quadruplexes in live cells2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 5, p. e27-e27Article in journal (Refereed)
    Abstract [en]

    Guanine (G)-rich nucleic acids are prone to assemble into four-stranded structures, so-called G-quadruplexes. Abnormal GGGGCC repeat elongations, and in particular their folding states, are associated with amyotrophic lateral sclerosis and frontotemporal dementia. Due to methodological constraints however, most studies of G quadruplex structures are restricted to in vitro conditions. Evidence of how GGGGCC repeats form into G-quadruplexes in vivo is sparse. We devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. Thereby, folding states of cyanine-labeled RNA, and in particular G-quadruplexes, can be identified in a sensitive manner. The isomerization kinetics, monitored via fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA with GGGGCC repeats in aqueous solution using fluorescence correlation spectroscopy and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with laser excitation modulation characteristics, we could then detect folding states of fluorescently tagged RNA introduced into live cells. 

  • 21.
    Krahn, Natalie
    et al.
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
    Zhang, Jingji
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
    Melnikov, Sergey V.
    Biosciences Institute, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
    Tharp, Jeffery M.
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
    Villa, Alessandra
    KTH, School of Electrical Engineering and Computer Science (EECS), Centres, Centre for High Performance Computing, PDC.
    Patel, Armaan
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
    Howard, Rebecca J.
    Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, SE-171 65, Sweden.
    Gabir, Haben
    Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
    Patel, Trushar R.
    Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, AB T1K 2E1, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada; Department of Microbiology, Immunology & Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
    Stetefeld, Jörg
    Department of Chemistry, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
    Puglisi, Joseph
    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
    Söll, Dieter
    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Chemistry, Yale University, New Haven, CT 06520, USA.
    tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut2024In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 52, no 2, p. 513-524Article in journal (Refereed)
    Abstract [en]

    Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

  • 22.
    Käll, Lukas
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, SeRC - Swedish e-Science Research Centre. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Krogh, Anders
    Sonnhammer, Erik L. L.
    Advantages of combined transmembrane topology and signal peptide prediction - the Phobius web server2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no Web Server issue, 1, p. W429-W432Article in journal (Refereed)
    Abstract [en]

    When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of predictions. Applying these methods to five complete proteomes, we found that 30-65% of all predicted signal peptides and 25-35% of all predicted transmembrane topologies overlap. This impairs predictions of 5-10% of the proteome, hence this is an important issue in protein annotation. To address this problem, we previously designed a hidden Markov model, Phobius, that combines transmembrane topology and signal peptide predictions. The method makes an optimal choice between transmembrane segments and signal peptides, and also allows constrained and homology-enriched predictions. We here present a web interface (http://phobius.cgb.ki.se and http://phobius.binf.ku.dk) to access Phobius.

  • 23.
    Lebrigand, Kevin
    et al.
    Univ Cote DAzur, Inst Pharmacol Mol & Cellulaire, CNRS, F-06560 Sophia Antipolis, France..
    Bergenstråhle, Joseph
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Thrane, Kim
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mollbrink, Annelie
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Meletis, Konstantinos
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden..
    Barbry, Pascal
    Univ Cote DAzur, Inst Pharmacol Mol & Cellulaire, CNRS, F-06560 Sophia Antipolis, France..
    Waldmann, Rainer
    Univ Cote DAzur, Inst Pharmacol Mol & Cellulaire, CNRS, F-06560 Sophia Antipolis, France..
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    The spatial landscape of gene expression isoforms in tissue sections2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 8Article in journal (Refereed)
    Abstract [en]

    In situ capturing technologies add tissue context to gene expression data, with the potential of providing a greater understanding of complex biological systems. However, splicing variants and full-length sequence heterogeneity cannot be characterized at spatial resolution with current transcriptome profiling methods. To that end, we introduce spatial isoform transcriptomics (SiT), an explorative method for characterizing spatial isoform variation and sequence heterogeneity using long-read sequencing. We show in mouse brain how SiT can be used to profile isoform expression and sequence heterogeneity in different areas of the tissue. SiT reveals regional isoform switching of Plp1 gene between different layers of the olfactory bulb, and the use of external single-cell data allows the nomination of cell types expressing each isoform. Furthermore, SiT identifies differential isoform usage for several major genes implicated in brain function (Snap25, Bin1, Gnas) that are independently validated by in situ sequencing. SiT also provides for the first time an in-depth A-to-I RNA editing map of the adult mouse brain. Data exploration can be performed through an online resource, where isoform expression and RNA editing can be visualized in a spatial context.

  • 24.
    Lee, Sunjae
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Korea Adv Inst Sci & Technol, South Korea.
    Mardinoglu, Adil
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers, Sweden.
    Zhang, Cheng
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lee, Doheon
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers, Sweden.
    Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 12, p. 5529-5539Article in journal (Refereed)
    Abstract [en]

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies.

  • 25.
    Lee, Sunjae
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Arif, Muhammad
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Liu, Zhengtao
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Benfeitas, Rui
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bidkhori, Gholamreza
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Deshmukh, Sumit
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Shobky, Mohamed AI
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lovric, Alen
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Boren, Jan
    Nielsen, Jens
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    TCSBN: a database of tissue and cancer specific biological networks2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no D1, p. D595-D600Article in journal (Refereed)
    Abstract [en]

    Biological networks provide new opportunities for understanding the cellular biology in both health and disease states. We generated tissue specific integrated networks (INs) for liver, muscle and adipose tissues by integratingmetabolic, regulatory and protein-protein interaction networks. We also generated human co-expression networks (CNs) for 46 normal tissues and 17 cancers to explore the functional relationships between genes as well as their relationships with biological functions, and investigate the overlap between functional and physical interactions provided by CNs and INs, respectively. These networks can be employed in the analysis of omics data, provide detailed insight into disease mechanisms by identifying the key biological components and eventually can be used in the development of efficient treatment strategies. Moreover, comparative analysis of the networks may allow for the identification of tissue-specific targets that can be used in the development of drugs with the minimum toxic effect to other human tissues. These context-specific INs and CNs are presented in an interactive website http://inetmodels.com without any limitation.

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  • 26.
    Lindahl, Erik
    et al.
    Stockholm University.
    Azuara, Cyril
    Institut Pasteur, Paris France.
    Koehl, Patrice
    Institut Pasteur, Paris France.
    Delarue, Marc
    NOMAD-Ref: visualization, deformation and refinement of macromolecular structures based on all-atom normal mode analysis2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no SI, p. W52-W56Article in journal (Refereed)
    Abstract [en]

    Normal mode analysis (NMA) is an efficient way to study collective motions in biomolecules that bypasses the computational costs and many limitations associated with full dynamics simulations. The NOMAD-Ref web server presented here provides tools for online calculation of the normal modes of large molecules (up to 100,000 atoms) maintaining a full all-atom representation of their structures, as well as access to a number of programs that utilize these collective motions for deformation and refinement of biomolecular structures. Applications include the generation of sets of decoys with correct stereochemistry but arbitrary large amplitude movements, the quantification of the overlap between alternative conformations of a molecule, refinement of structures against experimental data, such as X-ray diffraction structure factors or Cryo-EM maps and optimization of docked complexes by modeling receptor/ligand flexibility through normal mode motions. The server can be accessed at the URL http://lorentz.immstr.pasteur.fr/nomad-ref.php.

  • 27.
    Lindahl, Erik
    et al.
    Institut Pasteur, Paris, France.
    Delarue, Marc
    Institut Pasteur, Paris, France.
    Refinement of docked protein-ligand and protein-DNA structures using low frequency normal mode amplitude optimization2005In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 14, p. 4496-4506Article in journal (Refereed)
    Abstract [en]

    Prediction of structural changes resulting from complex formation, both in ligands and receptors, is an important and unsolved problem in structural biology. In this work, we use all-atom normal modes calculated with the Elastic Network Model as a basis set to model structural flexibility during formation of macromolecular complexes and refine the non-bonded intermolecular energy between the two partners (protein-ligand or protein-DNA) along 5-10 of the lowest frequency normal mode directions. The method handles motions unrelated to the docking transparently by first applying the modes that improve non-bonded energy most and optionally restraining amplitudes; in addition, the method can correct small errors in the ligand position when the first six rigid-body modes are switched on. For a test set of six protein receptors that show an open-to-close transition when binding small ligands, our refinement scheme reduces the protein coordinate cRMS by 0.3-3.2 A. For two test cases of DNA structures interacting with proteins, the program correctly refines the docked B-DNA starting form into the expected bent DNA, reducing the DNA cRMS from 8.4 to 4.8 A and from 8.7 to 5.4 A, respectively. A public web server implementation of the refinement method is available at http://lorentz.immstr.pasteur.fr.

  • 28.
    Lundqvist, Magnus
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Edfors, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hallström, Björn M
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Holmberg, Anders
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, article id e49Article in journal (Refereed)
    Abstract [en]

    We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.

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  • 29.
    Maaskola, Jonas
    et al.
    Max-Delbrück-Center for Molecular Medicine, Germany.
    Rajewsky, Nikolaus
    Binding site discovery from nucleic acid sequences by discriminative learning of hidden Markov models2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 21, p. 12995-13011Article in journal (Refereed)
    Abstract [en]

    We present a discriminative learning method for pattern discovery of binding sites in nucleic acid sequences based on hidden Markov models. Sets of positive and negative example sequences are mined for sequence motifs whose occurrence frequency varies between the sets. The method offers several objective functions, but we concentrate on mutual information of condition and motif occurrence. We perform a systematic comparison of our method and numerous published motif-finding tools. Our method achieves the highest motif discovery performance, while being faster than most published methods. We present case studies of data from various technologies, including ChIP-Seq, RIP-Chip and PAR-CLIP, of embryonic stem cell transcription factors and of RNA-binding proteins, demonstrating practicality and utility of the method. For the alternative splicing factor RBM10, our analysis finds motifs known to be splicing-relevant. The motif discovery method is implemented in the free software package Discrover. It is applicable to genome- and transcriptome-scale data, makes use of available repeat experiments and aside from binary contrasts also more complex data configurations can be utilized.

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  • 30. Maes, Alexandre
    et al.
    Céline, Gracia
    Innocenti, Nicolas
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST).
    Zhang, Kaiyang
    Aalto University, Finland .
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST).
    Hajnsdorf, Eliane
    Landscape of RNA polyadenylation in E. coli2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
    Abstract [en]

    Polyadenylation is involved in degradation and quality control of bacterial RNAs. We used a combination of 5’-tagRACE and RNA-seq to analyse the total RNA content from wild-type strain and from mutant deficient for poly(A)polymerase. We determined that 157 mRNAs were affected as well as non-coding transcripts, up- and downregulated in the mutant when compared to the wild-type strain. Antisense RNAs were also detected and differentially affected by polyadenylation.

    Our results clearly reveal a correlation between the RNA folding energy and the requirement of polyadenylation to achieve the RNA decay. A new algorithm was developed to detect in both strains posttranscriptional modifications based on unmappable 3’-ends to analyse their position and composition. Therefore, any RNA 3'-end can be polyadenylated addressing them to the exoribonucleolytic machinery which is essential to degrade structured RNAs. Importantly, poly(A)polymerase was also upregulating the expression of genes related with the entire FliA regulon and numerous membrane transporters while downregulating the expression of the antigen 43 (flu), numerous sRNAs, antisense transcripts, REP sequences with the accumulation of numerous RNA fragments resulting from the processing of entire transcripts. Altogether we show here that polyadenylation has a broader spectrum of action than was suspected until now.

  • 31. Milani, Lili
    et al.
    Gupta, Manu
    Andersen, Malin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Dhar, Sumeer
    Fryknäs, Mårten
    Isaksson, Anders
    Larsson, Rolf
    Syvänen, Ann-Christine
    Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5, p. E34-Article in journal (Refereed)
    Abstract [en]

    Using the relative expression levels of two SNIP alleles of a gene in the same sample is an effective approach for identifying cis-acting regulatory SNPs (rSNPs). In the current study, we established a process for systematic screening for cis-acting rSNPs using experimental detection of Al as an initial approach. We selected 160 expressed candidate genes that are involved in cancer and anticancer drug resistance for analysis of All in a panel of cell lines that represent different types of cancers and have been well characterized for their response patterns against anticancer drugs. Of these genes, 60 contained heterozygous SNPs in their coding regions, and 41 of the genes displayed imbalanced expression of the two cSNP alleles. Genes that displayed Al were subjected to bioinformatics-assisted identification of rSNPs that alter the strength of transcription factor binding. rSNPs in 15 genes were subjected to electrophoretic mobility shift assay, and in eight of these genes (APC, BCL2, CCND2, MLH1, PARP1, SLIT2, YES1, XRCC1) we identified differential protein binding from a nuclear extract between the SNIP alleles. The screening process allowed us to zoom in from 160 candidate genes to eight genes that may contain functional rSNPs in their promoter regions.

  • 32. Nookaew, Intawat
    et al.
    Papini, Marta
    Pornputtapong, Natapol
    Scalcinati, Gionata
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Matthias
    KTH, School of Biotechnology (BIO), Proteomics.
    Nielsen, Jens
    A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, p. 10084-10097Article in journal (Refereed)
    Abstract [en]

    RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated with the Illumina platform, and to perform a cross-platform comparison based on the results obtained through Affymetrix microarray. As a case study for our work we, used the Saccharomyces cerevisiae strain CEN.PK 113-7D, grown under two different conditions (batch and chemostat). Here, we asses the influence of genetic variation on the estimation of gene expression level using three different aligners for read-mapping (Gsnap, Stampy and TopHat) on S288c genome, the capabilities of five different statistical methods to detect differential gene expression (baySeq, Cuffdiff, DESeq, edgeR and NOISeq) and we explored the consistency between RNA-seq analysis using reference genome and de novo assembly approach. High reproducibility among biological replicates (correlation >= 0.99) and high consistency between the two platforms for analysis of gene expression levels (correlation >= 0.91) are reported. The results from differential gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays) for gene expression analysis and addresses the contribution of the different steps involved in the analysis of RNA-seq data.

  • 33. O'Meara, D.
    et al.
    Ahmadian, Afshin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments (pre-2005), Biotechnology.
    SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays2002In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 30, no 15Article in journal (Refereed)
    Abstract [en]

    This study reports the development of a microarray-based allele-specific extension method for typing of single nucleotide polymorphisms (SNPs). The use of allele-specific primers has been employed previously to identify single base variation but it is acknowledged that certain mismatches are not refractory to extension. Here we have overcome this limitation by introducing apyrase, a nucleotide-degrading enzyme, to the extension reaction. We have show previously that DNA polymerases exhibit slower reaction kinetics when extending a mismatched primer compared with a matched primer. This kinetic difference is exploited in the apyrase-mediated allele-specific extension (AMASE) assay, allowing incorporation of nucleotides when the reaction kinetics are fast but degrading the nucleotides before extension when the reaction kinetics are slow. Here we show that five homozygous variants (14% of the total number of variants) that were incorrectly scored in the absence of apyrase were correctly typed when apyrase was included in the extension reaction. AMASE was performed in situ on the oligonucleotide microarrarys using fluorescent nucleotides to type 10 SNPs and two indels in 17 individuals generating approximately 200 genotypes. Cluster analysis of these data shows three distinct clusters with clear-cut boundaries. We conclude that SNP typing on oligonucleotide microarrays by AMASE is an efficient, rapid and accurate technique for large-scale genotyping.

  • 34.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO).
    Lindskog, Mats
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO).
    Tri-nucleotide Threading for parallel amplification of minute amounts of genomic DNA2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 6, p. 9-Article in journal (Refereed)
    Abstract [en]

    Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

  • 35.
    Redin, David
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute (KI), Sweden.
    He, Mengxiao
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 13, article id e125Article in journal (Refereed)
    Abstract [en]

    Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.

  • 36.
    Reimegård, Johan
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Kundu, Snehangshu
    Pendle, Ali
    Irish, Vivian F
    Shaw, Peter
    Nakayama, Naomi
    Sundström, Jens F
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
    Abstract [en]

    Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.

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  • 37.
    Sandberg, Julia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus K
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Flow cytometry for enrichment and titration in massively parallel DNA sequencing2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 8Article in journal (Refereed)
    Abstract [en]

    Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.

  • 38. Sirk, S. J.
    et al.
    Gaj, T.
    Jonsson, Andreas
    KTH, School of Biotechnology (BIO), Protein Technology. Scripps Research Institute, United States .
    Mercer, A. C.
    Barbas, C. F.
    Expanding the zinc-finger recombinase repertoire: Directed evolution and mutational analysis of serine recombinase specificity determinants2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 7, p. 4755-4766Article in journal (Refereed)
    Abstract [en]

    The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys 2-His2 zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications the flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine recombinase catalytic specificity has been impeded by conserved base requirements within each recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity these results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.

  • 39. Ström, Cecilia E.
    et al.
    Johansson, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics.
    Erixon, Klaus
    Helleday, Thomas
    Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3166-3175Article in journal (Refereed)
    Abstract [en]

    Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

  • 40.
    Unneberg, Per
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Wennborg, Anders
    Larsson, Magnus
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Transcript identification by analysis of short sequence tags-influence of tag length, restriction site and transcript database2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 8, p. 2217-2226Article in journal (Refereed)
    Abstract [en]

    There exist a number of gene expression profiling techniques that utilize restriction enzymes for generation of short expressed sequence tags. We have studied how the choice of restriction enzyme influences various characteristics of tags generated in an experiment. We have also investigated various aspects of in silico transcript identification that these profiling methods rely on. First, analysis of 14 248 mRNA sequences derived from the RefSeq transcript database showed that 1-30% of the sequences lack a given restriction enzyme recognition site. Moreover, 1-5% of the transcripts have recognition sites located less than 10 bases from the poly(A) tail. The uniqueness of 10 bp tags lies in the range 90-95%, which increases only slightly with longer tags, due to the existence of closely related transcripts. Furthermore, 3-30% of upstream 10 bp tags are identical to 3′ tags, introducing a risk of misclassification if upstream tags are present in a sample. Second, we found that a sequence length of 16-17 bp, including the recognition site, is sufficient for unique transcript identification by BLAST based sequence alignment to the UniGene Human non-redundant database. Third, we constructed a tag-to-gene mapping for UniGene and compared it to an existing mapping database. The mappings agreed to 79-83%, where the selection of representative sequences in the UniGene clusters is the main cause of the disagreement. The results of this study may serve to improve the interpretation of sequence-based expression studies and the design of hybridization arrays, by identifying short tags that have a high reliability and separating them from tags that carry an inherent ambiguity in their capacity to discriminate between genes. To this end, supplementary information in the form of a web companion to this paper is located at http://biobase.biotech.kth.se/tagseq.

  • 41.
    Wallner, Björn
    et al.
    Department of Biochemistry, University of Washington.
    Larsson, Per
    Department of Biochemistry, University of Washington.
    Elofsson, Arne
    Department of Biochemistry, University of Washington.
    Pcons.net: Protein structure prediction meta server2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no Web Server issue, p. 369-374Article in journal (Refereed)
    Abstract [en]

    The Pcons.net Meta Server (http://pcons.net) provides improved automated tools for protein structure prediction and analysis using consensus. It essentially implements all the steps necessary to produce a high quality model of a protein. The whole process is fully automated and a potential user only submits the protein sequence. For PSI-BLAST detectable targets, an accurate model is generated within minutes of submission. For more difficult targets the sequence is automatically submitted to publicly available fold-recognition servers that use more advanced approaches to find distant structural homologs. The results from these servers are analyzed and assessed for structural correctness using Pcons and ProQ; and the user is presented with a ranked list of possible models. In addition, if the protein sequence contains more than one domain, these are automatically parsed out and resubmitted to the server as individual queries.

  • 42.
    Wang, Zhijia
    et al.
    Department of Biochemistry and Developmental Biology, University of Helsinki, Helsinki FIN-00014, Finland.
    Himanen, Samu V.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Haikala, Heidi M.
    Translational Immunology Research Program (TRIMM), Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki FIN-00014, Finland; iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki, Helsinki FIN-00014, Finland.
    Friedel, Caroline C.
    Institute of Informatics, Ludwig-Maximilians-Universität München, 80333 Munich, Germany.
    Vihervaara, Anniina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Barborič, Matjaž
    Department of Biochemistry and Developmental Biology, University of Helsinki, Helsinki FIN-00014, Finland.
    Inhibition of CDK12 elevates cancer cell dependence on P-TEFb by stimulation of RNA polymerase II pause release2023In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 51, no 20, p. 10970-10991Article in journal (Refereed)
    Abstract [en]

    P-TEFb and CDK12 facilitate transcriptional elongation by RNA polymerase II. Given the prominence of both kinases in cancer, gaining a better understanding of their interplay could inform the design of novel anti-cancer strategies. While down-regulation of DNA repair genes in CDK12-targeted cancer cells is being explored therapeutically, little is known about mechanisms and significance of transcriptional induction upon inhibition of CDK12. We show that selective targeting of CDK12 in colon cancer-derived cells activates P-TEFb via its release from the inhibitory 7SK snRNP. In turn, P-TEFb stimulates Pol II pause release at thousands of genes, most of which become newly dependent on P-TEFb. Amongst the induced genes are those stimulated by hallmark pathways in cancer, including p53 and NF-κB. Consequently, CDK12-inhibited cancer cells exhibit hypersensitivity to inhibitors of P-TEFb. While blocking P-TEFb triggers their apoptosis in a p53-dependent manner, it impedes cell proliferation irrespective of p53 by preventing induction of genes downstream of the DNA damage-induced NF-κB signaling. In summary, stimulation of Pol II pause release at the signal-responsive genes underlies the functional dependence of CDK12-inhibited cancer cells on P-TEFb. Our study establishes the mechanistic underpinning for combinatorial targeting of CDK12 with either P-TEFb or the induced oncogenic pathways in cancer.

  • 43.
    Werner, Arne
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 3, p. e17-Article in journal (Refereed)
    Abstract [en]

    Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of high-resolution structures, hydrodynamic radius and diffusion coefficient of evolutionary conserved RNA single strands (ssRNA) were calculated. The diffusion coefficients D of 17-174 nucleotides (nt) containing ssRNA depended on the number of nucleotides N with D = 4.56 x 10(-10) N-0.39 m(2) s(-1). The hydrodynamic radius R-H depended on N with R-H = 5.00 x 10(-10) N-0.38 m. An average ratio of the radius of gyration and the hydrodynamic radius of 0.98 +/- 0.08 was calculated in solution. The empirical law was tested by in solution measured hydrodynamic radii and radii of gyration and was found to be highly consistent with experimental data of evolutionary conserved ssRNA. Furthermore, the hydrodynamic behaviour of several evolutionary unevolved ribonucleic acids could be predicted. Based on atom-level shell-modelling of high-resolution structures and experimental hydrodynamic data, empirical models are proposed, which enable to predict the translational diffusion coefficient and molecular size of short RNA single strands solely on the basis of the polymer size.

  • 44. Wiegand, Stephan
    et al.
    Meier, Doreen
    Seehafer, Carsten
    Malicki, Marek
    Hofmann, Patrick
    Schmith, Anika
    Winckler, Thomas
    Foeldesi, Balint
    Boesler, Benjamin
    Nellen, Wolfgang
    Reimegård, Johan
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Avesson, Lotta
    Söderbom, Fredrik
    Hammann, Christian
    The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 5, p. 3330-3345Article in journal (Refereed)
    Abstract [en]

    Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC(-) strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC(-) strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5' and 3' directions.

  • 45. Yu, Nancy Yiu-Lin
    et al.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Kawaji, Hideya
    Carninci, Piero
    Forrest, Alistair R. R.
    Hayashizaki, Yoshihide
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Daub, Carsten O.
    Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 14, p. 6787-6798Article in journal (Refereed)
    Abstract [en]

    Understanding the normal state of human tissue transcriptome profiles is essential for recognizing tissue disease states and identifying disease markers. Recently, the Human Protein Atlas and the FANTOM5 consortium have each published extensive transcriptome data for human samples using Illumina-sequenced RNA-Seq and Heliscope-sequenced CAGE. Here, we report on the first large-scale complex tissue transcriptome comparison between full-length versus 5'-capped mRNA sequencing data. Overall gene expression correlation was high between the 22 corresponding tissues analyzed (R > 0.8). For genes ubiquitously expressed across all tissues, the two data sets showed high genome-wide correlation (91% agreement), with differences observed for a small number of individual genes indicating the need to update their gene models. Among the identified single-tissue enriched genes, up to 75% showed consensus of 7-fold enrichment in the same tissue in both methods, while another 17% exhibited multiple tissue enrichment and/or high expression variety in the other data set, likely dependent on the cell type proportions included in each tissue sample. Our results show that RNA-Seq and CAGE tissue transcriptome data sets are highly complementary for improving gene model annotations and highlight biological complexities within tissue transcriptomes. Furthermore, integration with image-based protein expression data is highly advantageous for understanding expression specificities for many genes.

  • 46. Zheng, Z.
    et al.
    Advani, A.
    Melefors, O.
    Glavas, S.
    Nordström, H.
    Ye, W.
    Engstrand, L.
    Andersson, Anders
    Swedish Institute for Infectious Disease Control, Solna, Sweden. Limnology/Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University .
    Titration-free massively parallel pyrosequencing using trace amounts of starting material2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 13Article in journal (Refereed)
    Abstract [en]

    Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable.

  • 47. Zhu, Y.
    et al.
    Engström, P. G.
    Tellgren-Roth, C.
    Baudo, C. D.
    Kennell, J. C.
    Sun, S.
    Billmyre, R. B.
    Schröder, M. S.
    Andersson, A.
    Holm, T.
    Sigurgeirsson, Benjamin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wu, G.
    Sankaranarayanan, S. R.
    Siddharthan, R.
    Sanyal, K.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystedt, B.
    Boekhout, T.
    Dawson, T.L., Jr.
    Heitman, J.
    Scheynius, A.
    Lehtiö, J.
    Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 5, p. 2629-2643Article in journal (Refereed)
    Abstract [en]

    Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.

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