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  • 1.
    Ajawatanawong, Pravech
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Atkinson, Gemma C.
    Watson-Haigh, Nathan S.
    MacKenzie, Bryony
    Baldauf, Sandra L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    SeqFIRE: a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no W1, p. W340-W347Article in journal (Refereed)
    Abstract [en]

    Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.

  • 2.
    Ameur, Adam
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Rada-Iglesias, Alvaro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Identification of candidate regulatory SNPs by combination of transcription-factor-binding site prediction, SNP genotyping and haploChIP2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 12, p. e85-Article in journal (Refereed)
    Abstract [en]

    Disease-associated SNPs detected in large-scale association studies are   frequently located in non-coding genomic regions, suggesting that they may be involved in transcriptional regulation. Here we describe a new strategy for detecting regulatory SNPs (rSNPs), by combining   computational and experimental approaches. Whole genome ChIP-chip data   for USF1 was analyzed using a novel motif finding algorithm called   BCRANK. 1754 binding sites were identified and 140 candidate rSNPs were   found in the predicted sites. For validating their regulatory function,   seven SNPs found to be heterozygous in at least one of four human cell   samples were investigated by ChIP and sequence analysis (haploChIP). In   four of five cases where the SNP was predicted to affect binding, USF1   was preferentially bound to the allele containing the consensus motif.   Allelic differences in binding for other proteins and histone marks   further reinforced the SNPs regulatory potential. Moreover, for one of   these SNPs, H3K36me3 and POLR2A levels at neighboring heterozygous SNPs   indicated effects on transcription. Our strategy, which is entirely   based on in vivo data for both the prediction and validation steps, can   identify individual binding sites at base pair resolution and predict   rSNPs. Overall, this approach can help to pinpoint the causative SNPs   in complex disorders where the associated haplotypes are located in regulatory regions. Availability: BCRANK is available from Bioconductor  (http://www.bioconductor.org).

  • 3. Andersen, Felicie F.
    et al.
    Knudsen, Bjarne
    Oliveira, Cristiano Luis Pinto
    Frøhlich, Rikke F.
    Krüger, Dinna
    Bungert, Jörg
    Agbandje-McKenna, Mavis
    McKenna, Robert
    Juul, Sissel
    Veigaard, Christopher
    Koch, Jørn
    Rubinstein, John L.
    Guldbrandtsen, Bernt
    Hede, Marianne S.
    Karlsson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Andersen, Anni H.
    Pedersen, Jan Skov
    Knudsen, Birgitta R.
    Assembly and structural analysis of a covalently closed nano-scale DNA cage2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 4, p. 1113-1119Article in journal (Refereed)
    Abstract [en]

    The inherent properties of DNA as a stable polymer with unique affinity for partner mols. detd. by the specific Watson-Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnol. Here we present the design, construction and structural anal. of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helixes that assembles from eight oligonucleotides with a yield of .apprx.30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-mols. to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

  • 4.
    Antson, D.O.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    PCR-generated padlock probes detect single nucleotide variation in genomic DNA2000In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 28, no 12, p. E58-Article in journal (Refereed)
    Abstract [en]

    Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5'- and 3'-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.

  • 5.
    Ardell, David H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Siv G. E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    TFAM detects co-evolution of tRNA identity rules with lateral transfer of histidyl-tRNA sythetase2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 3, p. 893-904Article in journal (Refereed)
    Abstract [en]

    We present TFAM, an automated, statistical method to classify the identity of tRNAs. TFAM, currently optimized for bacteria, classifies initiator tRNAs and predicts the charging identity of both typical and atypical tRNAs such as suppressors with high confidence. We show statistical evidence for extensive variation in tRNA identity determinants among bacterial genomes due to variation in overall tDNA base content. With TFAM we have detected the first case of eukaryotic-like tRNA identity rules in bacteria. An alpha-proteobacterial clade encompassing Rhizobiales, Caulobacter crescentus and Silicibacter pomeroyi, unlike a sister clade containing the Rickettsiales, Zymomonas mobilis and Gluconobacter oxydans, uses the eukaryotic identity element A73 instead of the highly conserved prokaryotic element C73. We confirm divergence of bacterial histidylation rules by demonstrating perfect covariation of alpha-proteobacterial tRNA(His) acceptor stems and residues in the motif IIb tRNA-binding pocket of their histidyl-tRNA synthetases (HisRS). Phylogenomic analysis supports lateral transfer of a eukaryotic-like HisRS into the alpha-proteobacteria followed by in situ adaptation of the bacterial tDNA(His) and identity rule divergence. Our results demonstrate that TFAM is an effective tool for the bioinformatics, comparative genomics and evolutionary study of tRNA identity.

  • 6.
    Ardui, Simon
    et al.
    Katholieke Univ Leuven, Dept Human Genet, B-3000 Leuven, Belgium..
    Ameur, Adam
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Monash Univ, Sch Publ Hlth & Prevent Med, Melbourne, Vic, Australia..
    Vermeesch, Joris R.
    Katholieke Univ Leuven, Dept Human Genet, B-3000 Leuven, Belgium..
    Hestand, Matthew S.
    Katholieke Univ Leuven, Dept Human Genet, B-3000 Leuven, Belgium.;Vrije Univ Amsterdam, Med Ctr, Dept Clin Genet, Amsterdam, Netherlands.;Cincinnati Childrens Hosp Med Ctr, Div Human Genet, Cincinnati, OH 45229 USA..
    Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 5, p. 2159-2168Article in journal (Refereed)
    Abstract [en]

    Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule realtime (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.

  • 7. Aspegren, Anders
    et al.
    Hinas, Andrea
    Larsson, Pontus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Anders
    Söderbom, Fredrik
    Novel non-coding RNAs in Dictyostelium discoideum and their expression during development2004In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 32, no 15, p. 4646-4656Article in journal (Refereed)
    Abstract [en]

    The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.

  • 8.
    Banér, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, Anders
    Waldenström, Erik
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Parallel gene analysis with allele-specific padlock probes and tag microarrays2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 17, p. e103-Article in journal (Refereed)
    Abstract [en]

    Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.

  • 9.
    Belikov, Sergey
    et al.
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wrange, Orjan
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden..
    Quantification of transcription factor-DNA binding affinity in a living cell2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, p. 3045-3058Article in journal (Refereed)
    Abstract [en]

    The apparent dissociation constant (K-d) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [H-3]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent K-d of similar to 1 mu M and dramatically stimulated DNA binding by AR with an apparent K-d of similar to 0.13 mu M at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.

  • 10.
    Berglund, Ann-Charlotte
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Sjölund, Erik
    Östlund, Gabriel
    Sonnhammer, Erik L. L.
    InParanoid 6: eukaryotic ortholog clusters with inparalogs2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, p. D263-D266Article in journal (Refereed)
    Abstract [en]

    The InParanoid eukaryotic ortholog database (http://InParanoid.sbc.su.se/) has been updated to version 6 and is now based on 35 species. We collected all available complete eukaryotic proteomes and Escherichia coli, and calculated ortholog groups for all 595 species pairs using the InParanoid program. This resulted in 2 642 187 pairwise ortholog groups in total. The orthology-based species relations are presented in an orthophylogram. InParanoid clusters contain one or more orthologs from each of the two species. Multiple orthologs in the same species, i.e. inparalogs, result from gene duplications after the species divergence. A new InParanoid website has been developed which is optimized for speed both for users and for updating the system. The XML output format has been improved for efficient processing of the InParanoid ortholog clusters.

  • 11. Bergqvist, A
    et al.
    Nilsson, M
    Bondeson, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Magnusson, G
    Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.1990In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 18, no 9, p. 2715-20Article in journal (Refereed)
    Abstract [en]

    A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function.

  • 12.
    Bergqvist, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.1990In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
  • 13.
    Borg, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pavlov, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, p. 3264-3275Article in journal (Refereed)
    Abstract [en]

    The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.

  • 14.
    Bourges, Anais C.
    et al.
    Rensselaer Polytech Inst, Dept Biol Sci, Troy, NY 12180 USA.;Univ Montpellier, CNRS UMR5048, INSERM U1054, Ctr Biochim Struct, F-34000 Montpellier, France..
    Montaguth, Oscar E. Torres
    Katholieke Univ Leuven, Food Microbiol Lab, Dept Microbial & Mol Syst, B-3001 Leuven, Belgium..
    Ghosh, Anirban
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Katholieke Univ Leuven, Food Microbiol Lab, Dept Microbial & Mol Syst, B-3001 Leuven, Belgium.
    Tadesse, Wubishet M.
    Katholieke Univ Leuven, Food Microbiol Lab, Dept Microbial & Mol Syst, B-3001 Leuven, Belgium..
    Declerck, Nathalie
    Univ Montpellier, CNRS UMR5048, INSERM U1054, Ctr Biochim Struct, F-34000 Montpellier, France..
    Aertsen, Abram
    Katholieke Univ Leuven, Food Microbiol Lab, Dept Microbial & Mol Syst, B-3001 Leuven, Belgium..
    Royer, Catherine A.
    Rensselaer Polytech Inst, Dept Biol Sci, Troy, NY 12180 USA..
    High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 9, p. 5323-5332Article in journal (Refereed)
    Abstract [en]

    A sub-lethal hydrostatic pressure (HP) shock of similar to 100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M. HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M. HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M. HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M. HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.

  • 15. Bramsen, Jesper B.
    et al.
    Laursen, Maria B
    Nielsen, Anne F.
    Hansen, Thomas B.
    Bus, Claus
    Langkjær, Niels
    Babu, B. Ravindra
    Højland, Torben
    Abramov, Mikhail
    van Aerschot, Arthur
    Odadzic, Dalibor
    Smicius, Romualdas
    Haas, Jens
    Andree, Cordula
    Barman, Jharna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Wenska, Malgorzata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Srivastava, Puneet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Zhou, Chuanzheng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Honcharenko, Dmytro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Hess, Simone
    Müller, Elke
    Bobkov, Georgii V.
    Mikhailov, Sergey N.
    Fava, Eugenio
    Meyer, Thomas F.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Zerial, Marino
    Engels, Joachim W.
    Herdewijn, Piet
    Wengel, Jesper
    Kjems, Jørgen
    A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 9, p. 2867-2881Article in journal (Refereed)
    Abstract [en]

    The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

  • 16. Bramsen, Jesper B.
    et al.
    Pakula, Malgorzata M.
    Hansen, Thomas B.
    Bus, Claus
    Langkjaer, Niels
    Odadzic, Dalibor
    Smicius, Romualdas
    Wengel, Suzy L.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Engels, Joachim W.
    Herdewijn, Piet
    Wengel, Jesper
    Kjems, Jorgen
    A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 17, p. 5761-5773Article in journal (Refereed)
    Abstract [en]

    Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

  • 17. Brännvall, M
    et al.
    Mikkelsen, NE
    Kirsebom, Leif A
    Monitoring the structure of Escherichia coli RNase P RNA in the presence of various metal ion2001In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, p. 1426-1432Article in journal (Refereed)
  • 18.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Göransson, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Razzaghian, Hamidreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Öberg, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Single molecule analysis of combinatorial splicing2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 16, p. e163-Article in journal (Refereed)
    Abstract [en]

    Alternative splicing forms diverse mRNA isoform populations from a single ancestral pre-mRNA and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns into a library of binary strings of nucleic acid tags, encoding the exon composition of transcripts. The transcript abundance is registered by counts of individual molecules and individual exon inclusion patterns are represented as strings of binary data.

    The technique is illustrated by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits different genes to be analyzed in parallel and will be valuable for elucidating the complex effects of combinatorial splicing.

  • 19.
    Coppotelli, Giuseppe
    et al.
    Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
    Mughal, Nouman
    Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
    Callegari, Simone
    Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
    Sompallae, Ramakrishna
    Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA.
    Caja Puigsubira, Laia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Luijsterburg, Martijn S
    Department of Toxicogenetics, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands .
    Dantuma, Nico P
    Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Masucci, Maria G
    Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden.
    The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 5, p. 2950-2962Article in journal (Refereed)
    Abstract [en]

    Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

  • 20.
    Diamanti, Klev
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Umer, Husen M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Kruczyk, Marcin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Dabrowski, Michal J.
    Polish Acad Sci, Inst Comp Sci, PL-01248 Warsaw, Poland..
    Cavalli, Marco
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Komorowski, Jan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Polish Acad Sci, Inst Comp Sci, PL-01248 Warsaw, Poland..
    Maps of context-dependent putative regulatory regions and genomic signal interactions2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 19, p. 9110-9120Article in journal (Refereed)
    Abstract [en]

    Gene transcription is regulated mainly by transcription factors (TFs). ENCODE and Roadmap Epigenomics provide global binding profiles of TFs, which can be used to identify regulatory regions. To this end we implemented a method to systematically construct cell-type and species-specific maps of regulatory regions and TF-TF interactions. We illustrated the approach by developing maps for five human cell-lines and two other species. We detected similar to 144k putative regulatory regions among the human cell-lines, with the majority of them being similar to 300 bp. We found similar to 20k putative regulatory elements in the ENCODE heterochromatic domains suggesting a large regulatory potential in the regions presumed transcriptionally silent. Among the most significant TF interactions identified in the heterochromatic regions were CTCF and the cohesin complex, which is in agreement with previous reports. Finally, we investigated the enrichment of the obtained putative regulatory regions in the 3D chromatin domains. More than 90% of the regions were discovered in the 3D contacting domains. We found a significant enrichment of GWAS SNPs in the putative regulatory regions. These significant enrichments provide evidence that the regulatory regions play a crucial role in the genomic structural stability. Additionally, we generated maps of putative regulatory regions for prostate and colorectal cancer human cell-lines.

  • 21. Dierckx, Anke
    et al.
    Dinér, Peter
    University of Gothenburg.
    El-Sagheer, Afaf H
    Kumar, Joshi Dhruval
    Brown, Tom
    Grøtli, Morten
    Wilhelmsson, L Marcus
    Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA.2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 10, p. 4513-24Article in journal (Refereed)
    Abstract [en]

    To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (A(T)) and its photophysical characterization inside DNA. A(T) shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, A(T) shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that A(T) only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that A(T) shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, A(T) shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

  • 22.
    Ederth, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 2, p. e15-Article in journal (Refereed)
    Abstract [en]

    With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

  • 23.
    Elvers, Ingegerd
    et al.
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Johansson, Fredrik
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Djureinovic, Tatjana
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Lagerqvist, Ann
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Schultz, Niklas
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Stoimenov, Ivaylo
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Erikson, Klas
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    Helleday, Thomas
    Department of Genetics, Microbiology, and Toxicology, Stockholm University, S-10691, Stockholm, Sweden.
    CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Other academic)
  • 24.
    Enroth, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Andersson, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Bysani, Madhusudhan Reddy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Tuch, Brian
    De la Vega, Fransisco
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Nucleosome regulatory dynamics in response to TGF-beta treatment in HepG2 cells2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 11, p. 6921-6934Article in journal (Refereed)
  • 25.
    Enroth, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Andersson, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Bysani, Madhusudhan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Termén, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tuch, Brian B
    Applied Biosystems, part of Life Technologies, Foster City, CA 94404, USA.
    De La Vega, Francisco M
    Applied Biosystems, part of Life Technologies, Foster City, CA 94404, USA.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Institute of Computer Science, Polish Academy of Sciences, ul. Jana Kazimierza 5, 01-248 Warszawa, Poland.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nucleosome regulatory dynamics in response to TGF beta2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 11, p. 6921-6934Article in journal (Refereed)
    Abstract [en]

    Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44-78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.

  • 26.
    Ericsson, Olle
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Howell, MAthias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nong, Rachel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Reuter, Hendrik
    Hahn, Meinhard
    Stenberg, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A dual-tag microarray platform for high-performance nucleic acid and protein analyses2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 8, p. e45-Article in journal (Refereed)
    Abstract [en]

    DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.

  • 27.
    Esguerra, Mauricio
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Siretskiy, Alexey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bello, Xabier
    Hosp Clin Univ Santiago, Fdn Publ Galega Med Xenom, Santiago De Compostela 15706, Spain..
    Sallander, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    GPCR-ModSim: A comprehensive web based solution for modeling G-protein coupled receptors2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no W1, p. W455-W462Article in journal (Refereed)
    Abstract [en]

    GPCR-ModSim (http://open.gpcr-modsim.org) is a centralized and easy to use service dedicated to the structural modeling of G-protein Coupled Receptors (GPCRs). 3D molecular models can be generated from amino acid sequence by homology-modeling techniques, considering different receptor conformations. GPCR-ModSim includes a membrane insertion and molecular dynamics (MD) equilibration protocol, which can be used to refine the generated model or any GPCR structure uploaded to the server, including if desired non-protein elements such as orthosteric or allosteric ligands, structural waters or ions. We herein revise the main characteristics of GPCR-ModSim and present new functionalities. The templates used for homology modeling have been updated considering the latest structural data, with separate profile structural alignments built for inactive, partially-active and active groups of templates. We have also added the possibility to perform multiple-template homology modeling in a unique and flexible way. Finally, our new MD protocol considers a series of distance restraints derived from a recently identified conserved network of helical contacts, allowing for a smoother refinement of the generated models which is particularly advised when there is low homology to the available templates. GPCR- ModSim has been tested on the GPCR Dock 2013 competition with satisfactory results.

  • 28.
    Flores, Samuel Coulbourn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fast fitting to low resolution density maps: elucidating large-scale motions of the ribosome2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 2, p. e9-Article in journal (Refereed)
    Abstract [en]

    Determining the conformational rearrangements of large macromolecules is challenging experimentally and computationally. Case in point is the ribosome; it has been observed by high-resolution crystallography in several states, but many others are known only from low-resolution methods including cryoelectron microscopy. Combining these data into dynamical trajectories that may aid understanding of its largest-scale conformational changes has so far remained out of reach of computational methods. Most existing methods either model all atoms explicitly, resulting in often prohibitive cost, or use approximations that lose interesting structural and dynamical detail. In this work, I introduce Internal Coordinate Flexible Fitting, which uses full atomic forces and flexibility in limited regions of a model, capturing extensive conformational rearrangements at low cost. I use it to turn multiple low-resolution density maps, crystallographic structures and biochemical information into unified all-atoms trajectories of ribosomal translocation. Internal Coordinate Flexible Fitting is three orders of magnitude faster than the most comparable existing method.

  • 29.
    Freyhult, Eva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Moulton, Vincent
    Clote, Peter
    RNAbor: a web server for RNA structural neighbors2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no Suppl. S: Web Server issue, p. W305-W309Article in journal (Refereed)
    Abstract [en]

    RNAbor provides a new tool for researchers in the biological and related sciences to explore important aspects of RNA secondary structure and folding pathways. RNAbor computes statistics concerning delta-neighbors of a given input RNA sequence and structure (the structure can, for example, be the minimum free energy (MFE) structure). A delta-neighbor is a structure that differs from the input structure by exactly delta base pairs, that is, it can be obtained from the input structure by adding and/or removing exactly d base pairs. For each distance delta RNAbor computes the density of delta-neighbors, the number of delta-neighbors, and the MFE structure, or MFEd structure, among all delta-neighbors. RNAbor can be used to study possible folding pathways, to determine alternate low-energy structures, to predict potential nucleation sites and to explore structural neighbors of an intermediate, biologically active structure. The web server is available at http://bioinformatics.bc.edu/clotelab/RNAbor.

  • 30.
    Galli, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Oelrich, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Taussig, Michael J
    Andreasson, Ulrika
    Ortega-Paino, Eva
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    The Biobanking Analysis Resource Catalogue (BARCdb): a new research tool for the analysis of biobank samples.2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no D1, p. D1158-D1162Article in journal (Refereed)
    Abstract [en]

    We report the development of a new database of technology services and products for analysis of biobank samples in biomedical research. BARCdb, the Biobanking Analysis Resource Catalogue (http://www.barcdb.org), is a freely available web resource, listing expertise and molecular resource capabilities of research centres and biotechnology companies. The database is designed for researchers who require information on how to make best use of valuable biospecimens from biobanks and other sample collections, focusing on the choice of analytical techniques and the demands they make on the type of samples, pre-analytical sample preparation and amounts needed. BARCdb has been developed as part of the Swedish biobanking infrastructure (BBMRI.se), but now welcomes submissions from service providers throughout Europe. BARCdb can help match resource providers with potential users, stimulating transnational collaborations and ensuring compatibility of results from different labs. It can promote a more optimal use of European resources in general, both with respect to standard and more experimental technologies, as well as for valuable biobank samples. This article describes how information on service and reagent providers of relevant technologies is made available on BARCdb, and how this resource may contribute to strengthening biomedical research in academia and in the biotechnology and pharmaceutical industries.

  • 31. Glemarec, C
    et al.
    Kufel, J
    Földesi, A
    Maltseva, T
    Sandström, A
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Chattopadhyaya, J
    The NMR structure of 31-mer RNA domain of E. coli RNase P RNA using its non-uniformly deuterium labelled counterpart (the "NMR-window" concept)1996In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 24, no 11, p. 2022-2035Article in journal (Refereed)
    Abstract [en]

    The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli is reported. Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31 mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)] [for the 'NMR-window' concept see: Földesi,A. et al. (1992) Tetrahedron, 48, 9033; Foldesi,A. et al. (1993) J. Biochem. Biophys. Methods, 26, 1; Yamakage,S.-I. et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. et al. (1994) Nucleic Acids Res., 22, 1404; Földesi,A. et al. (1995) Tetrahedron, 51, 10065; Földesi,A. et al. (1996) Nucleic Acids Res., 24, 1187-1194]. 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of 13C and 15N labelling. 41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2). The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I. Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I. The loop I-stem II junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair.

  • 32. Gould, Cathryn M
    et al.
    Diella, Francesca
    Via, Allegra
    Puntervoll, Pål
    Gemünd, Christine
    Chabanis-Davidson, Sophie
    Michael, Sushama
    Sayadi, Ahmed
    Department of Biochemical Sciences, ‘Sapienza Universita’ di Roma, Rome, Italy.
    Bryne, Jan Christian
    Chica, Claudia
    Seiler, Markus
    Davey, Norman E
    Haslam, Niall
    Weatheritt, Robert J
    Budd, Aidan
    Hughes, Tim
    Pas, Jakub
    Rychlewski, Leszek
    Travé, Gilles
    Aasland, Rein
    Helmer-Citterich, Manuela
    Linding, Rune
    Gibson, Toby J
    ELM: the status of the 2010 eukaryotic linear motif resource.2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38Article in journal (Refereed)
    Abstract [en]

    Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.

  • 33. Griffith, Obi L.
    et al.
    Montgomery, Stephen B.
    Bernier, Bridget
    Chu, Bryan
    Kasaian, Katayoon
    Aerts, Stein
    Mahony, Shaun
    Sleumer, Monica C.
    Bilenky, Mikhail
    Haeussler, Maximilian
    Griffith, Malachi
    Gallo, Steven M.
    Giardine, Belinda
    Hooghe, Bart
    Van Loo, Peter
    Blanco, Enrique
    Ticoll, Amy
    Lithwick, Stuart
    Portales-Casamar, Elodie
    Donaldson, Ian J.
    Robertson, Gordon
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    De Bleser, Pieter
    Vlieghe, Dominique
    Halfon, Marc S.
    Wasserman, Wyeth
    Hardison, Ross
    Bergman, Casey M.
    Jones, Steven J. M.
    ORegAnno: an open-access community-driven resource for regulatory annotation2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no Database issue, p. D107-D113Article in journal (Refereed)
    Abstract [en]

    ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regulatory sequences for over 3853 genes and 465 transcription factors from 19 species. A new feature called the publication queue allows users to input relevant papers from scientific literature as targets for annotation. The queue contains 4438 gene regulation papers entered by experts and another 54 351 identified by text-mining methods. Users can enter or check out papers from the queue for manual curation using a series of user-friendly annotation pages. A typical record entry consists of species, sequence type, sequence, target gene, binding factor, experimental outcome and one or more lines of experimental evidence. An evidence ontology was developed to describe and categorize these experiments. Records are cross-referenced to Ensembl or Entrez gene identifiers, PubMed and dbSNP and can be visualized in the Ensembl or UCSC genome browsers. All data are freely available through search pages, XML data dumps or web services at: http://www.oreganno.org.

  • 34. Groth, P
    et al.
    Orta, ML
    Elvers, I
    Majumder, MM
    Lagerqvist, A
    Helleday, T
    Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 14, p. 6585-6594Article in journal (Refereed)
  • 35.
    Göransson, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Howell, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A single molecule array for digital targeted molecular analyses2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 1, p. e7-Article in journal (Refereed)
    Abstract [en]

    We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.

  • 36.
    Halvardson, Jonatan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zaghlool, Ammar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Feuk, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Exome RNA sequencing reveals rare and novel alternative transcripts2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 1, p. e6-Article in journal (Refereed)
    Abstract [en]

    RNA sequencing has become an important method to perform hypothesis-free characterization of global gene expression. One of the limitations of RNA sequencing is that most sequence reads represent highly expressed transcripts, whereas low level transcripts are challenging to detect. To combine the benefits of traditional expression arrays with the advantages of RNA sequencing, we have used whole exome enrichment prior to sequencing of total RNA. We show that whole exome capture can be successfully applied to cDNA to study the transcriptional landscape in human tissues. By introducing the exome enrichment step, we are able to identify transcripts present at very low levels, which are below the level of detection in conventional RNA sequencing. Although the enrichment increases the ability to detect presence of transcripts, it also lowers the accuracy of quantification of expression levels. Our results yield a large number of novel exons and splice isoforms, suggesting that conventional RNA sequencing methods only detect a small fraction of the full transcript diversity. We propose that whole exome enrichment of RNA is a suitable strategy for genome-wide discovery of novel transcripts, alternative splice variants and fusion genes.

  • 37.
    Hinas, Andrea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reimegård, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wagner, Gerhart H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nellen, Wolfgang
    Ambros, Victor R.
    Söderbom, Fredrik
    The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 20, p. 6714-6726Article in journal (Refereed)
    Abstract [en]

    Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 1826 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.

  • 38.
    Holmqvist, Erik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Reimegard, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 12, p. e122-Article in journal (Refereed)
    Abstract [en]

    We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.

  • 39. Hon, Chung-Chau
    et al.
    Weber, Christian
    Sismeiro, Odile
    Proux, Caroline
    Koutero, Mikael
    Deloger, Marc
    Das, Sarbashis
    Agrahari, Mridula
    Dillies, Marie-Agnes
    Jagla, Bernd
    Coppee, Jean-Yves
    Bhattacharya, Alok
    Guillen, Nancy
    Quantification of stochastic noise of splicing and polyadenylation in Entamoeba histolytica.2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 3, p. 1936-52Article in journal (Refereed)
    Abstract [en]

    Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited. Lastly, we revised the gene models and annotated their 3'UTR in AmoebaDB, providing valuable resources to the community.

  • 40.
    Hooper, Sean D
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Molecular Evolution.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Molecular Evolution.
    Gradients in nucleotide and codon usage along Escherichia coli genes2000In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 28, no 18, p. 3517-3523Article in journal (Refereed)
    Abstract [en]

    The usage of codons and nucleotide combinations varies along genes and systematic variation causes gradients in usage. We have studied such gradients of nucleotides and nucleotide combinations and their immediate context in Escherichia coli. To distinguish mutational and selectional effects, the genes were subdivided into three groups with different codon usage bias and the gradients of nucleotide usage were studied in each group. Some combinations that can be associated with a propensity for processivity errors show strong negative gradients that become weaker in genes with low codon bias, consistent with a selection on translational efficiency. One of the strongest gradients is for third position G, which shows a pervasive positive gradient in usage in most contexts of surrounding bases.

  • 41.
    Howell, W. Mathias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Grundberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Faryna, Marta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 7, p. e99-Article in journal (Refereed)
    Abstract [en]

    The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.

  • 42. Huang, Hsin-Ho
    et al.
    Camsund, Daniel
    Lindblad, Peter
    Heidorn, Thorsten
    Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
  • 43.
    Huang, Hsin-Ho
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Camsund, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Heidorn, Thorsten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 8, p. 2577-2593Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P-rnpB and variants of P-rbcL, and a version of the promoter P-trc with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

  • 44.
    Ingman, Max
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gyllensten, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences2006In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no Issue Database issue, 1, p. D749-D751Article in journal (Refereed)
    Abstract [en]

    The mitochondrial genome, contained in the subcellular mitochondrial network, encodes a small number of peptides pivotal for cellular energy production. Mitochondrial genes are highly polymorphic and cataloguing existing variation is of interest for medical scientists involved in the identification of mutations causing mitochondrial dysfunction, as well as for population genetics studies. Human Mitochondrial Genome Database (mtDB) (http://www.genpat.uu.se/mtDB) has provided a comprehensive database of complete human mitochondrial genomes since early 2000. At this time, owing to an increase in the number of published complete human mitochondrial genome sequences, it became necessary to provide a web-based database of human whole genome and complete coding region sequences. As of August 2005 this database contains 2104 sequences (1544 complete genome and 560 coding region) available to download or search for specific polymorphisms. Of special interest to medical researchers and population geneticists evaluating specific positions is a complete list of (currently 3311) mitochondrial polymorphisms among these sequences. Recent expansions in the capabilities of mtDB include a haplotype search function and the ability to identify and download sequences carrying particular variants.

  • 45.
    Isaksson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Stenberg, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dahl, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Thuresson, Ann-Charlotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Bondeson, Marie-Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    MLGA--a rapid and cost-efficient assay for gene copy-number analysis2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 17, p. e115-Article in journal (Refereed)
    Abstract [en]

    Structural variation is an important cause of genetic variation. Whole genome analysis techniques can efficiently identify copy-number variable regions but there is a need for targeted methods, to verify and accurately size variable regions, and to diagnose large sample cohorts. We have developed a technique based on multiplex amplification of size-coded selectively circularized genomic fragments, which is robust, cheaper and more rapid than current multiplex targeted copy-number assays.

  • 46.
    Ivanov, Maxim
    et al.
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Pharmacogenet, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    Kals, Mart
    Univ Tartu, Estonian Genome Ctr, Riia 23b, EE-51010 Tartu, Estonia.;Univ Tartu, Inst Math & Stat, J Liivi 2, EE-50409 Tartu, Estonia..
    Lauschke, Volker
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Pharmacogenet, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    Barragan, Isabel
    Karolinska Inst, Dept Physiol & Pharmacol, Grp Pharmacoepigenet, Von Eulers Vag 8 4, S-17177 Stockholm, Sweden..
    Ewels, Philip
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-10691 Stockholm, Sweden..
    Kaller, Max
    Royal Inst Technol, Sch Biotechnol, Div Gene Technol, Sci Life Lab, S-17121 Stockholm, Sweden..
    Axelsson, Tomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehtio, Janne
    Karolinska Inst, Dept Oncol Pathol, Sci Life Lab, Canc Prote Mass Spectrometry, S-17121 Stockholm, Sweden..
    Milani, Lili
    Univ Tartu, Estonian Genome Ctr, Riia 23b, EE-51010 Tartu, Estonia..
    Ingelman-Sundberg, Magnus
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Pharmacogenet, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    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.

  • 47.
    Johanson, Urban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    A new mutation in 16S rRNA of Escherichia coli conferring spectinomycin resistance1995In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 23, no 3, p. 464-466Article in journal (Refereed)
    Abstract [en]

    We report a novel mutation, Cl 066U in 16S rRNA whichwas selected for resistance to spectinomycin, anantibiotic which inhibits ribosomal translocation. Theminimal inhibitory concentration (MIC) of spectinomycindetermined for this mutant (15 pg/ml) is greaterthan with the wild-type plasmid (5 ig/ml) but lower thanwith the well known C1192U mutation (>80 pg/ml). TheCl 066U mutation also increases the cells sensitivity tofusidic acid, another antibiotic which inhibits translationat the translocation stage, whereas C1192U isunchanged relative to the wild type. We discuss whythe acquisition of resistance to one of these drugs isoften associated with hypersensitivity to the other.

  • 48.
    Johansson, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Isaksson, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sörqvist, Elin Falk
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Roos, F.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Stenberg, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sjöblom, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Edlund, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Fredriksson, S.
    Göransson Kultima, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Targeted resequencing of candidate genes using Selector Probes2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 2, p. e8-Article in journal (Refereed)
    Abstract [en]

    Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94 specificity and 98 coverage of the targeted region was achieved. Reproducibility between replicates was high (R 2=0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

  • 49. Johnson, Stephanie
    et al.
    Lindén, Martin
    Phillips, Rob
    Sequence dependence of transcription factor-mediated DNA looping2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 16, p. 7728-7738Article in journal (Refereed)
    Abstract [en]

    DNA is subject to large deformations in a wide range of biological processes. Two key examples illustrate how such deformations influence the readout of the genetic information: the sequestering of eukaryotic genes by nucleosomes and DNA looping in transcriptional regulation in both prokaryotes and eukaryotes. These kinds of regulatory problems are now becoming amenable to systematic quantitative dissection with a powerful dialogue between theory and experiment. Here, we use a single-molecule experiment in conjunction with a statistical mechanical model to test quantitative predictions for the behavior of DNA looping at short length scales and to determine how DNA sequence affects looping at these lengths. We calculate and measure how such looping depends upon four key biological parameters: the strength of the transcription factor binding sites, the concentration of the transcription factor, and the length and sequence of the DNA loop. Our studies lead to the surprising insight that sequences that are thought to be especially favorable for nucleosome formation because of high flexibility lead to no systematically detectable effect of sequence on looping, and begin to provide a picture of the distinctions between the short length scale mechanics of nucleosome formation and looping.

  • 50. Johnson, Stephanie
    et al.
    van de Meent, Jan-Willem
    Phillips, Rob
    Wiggins, Chris H
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 16, p. 10265-10277Article in journal (Refereed)
    Abstract [en]

    The bacterial transcription factor LacI loops DNA by binding to two separate locations on the DNA simultaneously. Despite being one of the best-studied model systems for transcriptional regulation, the number and conformations of loop structures accessible to LacI remain unclear, though the importance of multiple coexisting loops has been implicated in interactions between LacI and other cellular regulators of gene expression. To probe this issue, we have developed a new analysis method for tethered particle motion, a versatile and commonly used in vitro single-molecule technique. Our method, vbTPM, performs variational Bayesian inference in hidden Markov models. It learns the number of distinct states (i.e. DNA-protein conformations) directly from tethered particle motion data with better resolution than existing methods, while easily correcting for common experimental artifacts. Studying short (roughly 100 bp) LacI-mediated loops, we provide evidence for three distinct loop structures, more than previously reported in single-molecule studies. Moreover, our results confirm that changes in LacI conformation and DNA-binding topology both contribute to the repertoire of LacI-mediated loops formed in vitro, and provide qualitatively new input for models of looping and transcriptional regulation. We expect vbTPM to be broadly useful for probing complex protein-nucleic acid interactions.

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