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  • 151. Norling, Ameli
    et al.
    Hirschberg, Angelica Linden
    Iwarsson, Erik
    Persson, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wedell, Anna
    Barbaro, Michela
    Novel candidate genes for 46,XY gonadal dysgenesis identified by a customized 1 M array-CGH platform2013In: European Journal of Medical Genetics, ISSN 1769-7212, E-ISSN 1878-0849, Vol. 56, no 12, p. 661-668Article in journal (Refereed)
    Abstract [en]

    Half of all patients with a disorder of sex development (DSD) do not receive a specific molecular diagnosis. Comparative genomic hybridization (CGH) can detect copy number changes causing gene haploinsufficiency or over-expression that can lead to impaired gonadal development and gonadal DSD. The purpose of this study was to identify novel candidate genes for 46,XY gonadal dysgenesis (GD) using a customized 1 M array-CGH platform with whole-genome coverage and probe enrichment targeting 78 genes involved in sex development. Fourteen patients with 46,XY gonadal DSD were enrolled in the study. Nine individuals were analyzed by array CGH. All patients were included in a follow up sequencing study of candidate genes. Three novel candidate regions for 46,XY GD were identified in two patients. An interstitial duplication of the SUPT3H gene and a deletion of C2ORF80 were detected in a pair of affected siblings. Sequence analysis of these genes in all patients revealed no additional mutations. A large duplication highlighting PIP5K1B, PRKACG and FAM189A2 as candidates for 46, XY GD, were also detected. All five genes are expressed in testicular tissues, and one is shown to cause gonadal DSD in mice. However detailed functional information is lacking for these genes.

  • 152. Nyberg, L. K.
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, B.
    Westerlund, F.
    Assessing how cationic intercalators affect DNA using nanofluidic channels2013In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 42, p. S202-S202Article in journal (Other academic)
  • 153. Nyberg, Lena K.
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Berg, Johan
    Bergstrom, Johanna
    Fransson, Emelie
    Olsson, Linnea
    Persson, Moa
    Stalnacke, Antti
    Wigenius, Jens
    Tegenfeldt, Jonas O.
    Westerlund, Fredrik
    A single-step competitive binding assay for mapping of single DNA molecules2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 1, p. 404-408Article in journal (Refereed)
    Abstract [en]

    Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.

  • 154. Nyberg, Lena
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, Bjorn
    Westerlund, Fredrik
    Assessing How Cationic Intercalators Affect DNA using Nanofluidic Channels2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 165A-165AArticle in journal (Other academic)
  • 155. Nyberg, Lena
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, Bjorn
    Westerlund, Fredrik
    Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 19, p. e184-Article in journal (Refereed)
    Abstract [en]

    The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

  • 156. O'Donoghue, AnnMarie C.
    et al.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mechanisms: Chemical and computational probes of biological mechanism2014In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 21, p. VIII-XArticle in journal (Other academic)
  • 157.
    Olsson, Jan A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Nordström, Kurt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication2012In: Plasmid, ISSN 0147-619X, E-ISSN 1095-9890, Vol. 67, no 2, p. 191-198Article in journal (Refereed)
    Abstract [en]

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30 degrees C, but as growth temperatures were raised above 34 degrees C, the copy number of the plasmid increased to higher levels, and at 42 degrees C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42 degrees C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the RI population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  • 158.
    Orzechowski Westholm, Jakub
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Tronnersjö, Susanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nordberg, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Olsson, Ida
    Komorowski, Jan
    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 Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ronne, Hans
    Gis1 and Rph1 Regulate Glycerol and Acetate Metabolism in Glucose Depleted Yeast Cells2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2, p. e31577-Article in journal (Refereed)
    Abstract [en]

    Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl CoA metabolism are downregulated by Gis1.

  • 159. Ostberg, Linus J.
    et al.
    Persson, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hoog, Jan-Olov
    The mammalian alcohol dehydrogenase genome shows several gene duplications and gene losses resulting in a large set of different enzymes including pseudoenzymes2015In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 234, p. 80-84Article in journal (Refereed)
    Abstract [en]

    Mammalian alcohol dehydrogenase (ADH) is a protein family divided into six classes and the number of known family members is increasing rapidly. Several primate genomes are completely analyzed for the ADH region, where higher primates (human and hominoids) have seven genes of classes ADH1-ADH5. Within the group of non-hominoids apes there have been further duplications and species with more than the typical three isozymic forms for ADH1 are present. In contrast there are few completely analyzed ADH genomes in the non-primate group of mammals, where an additional class has been identified, ADH6, that has been lost during the evolution of primates. In this study 85 mammalian genomes with at least one ADH gene have been compiled. In total more than 500 ADH amino acid sequences were analyzed for patterns that distinguish the different classes. For ADH1-ADH4 intensive investigations have been performed both at the functional and at structural levels. However, a corresponding functional protein to the ADH5 gene, which is found in most ADH genomes, has never been detected. The same is true for ADH6, which is only present in non-primates. The entire mammalian ADH family shows a broad spectrum of gene duplications and gene losses where the numbers differ from six genes (most non-primate mammals) up to ten genes (vole). Included in these sets are examples of pseudogenes and pseudoenzymes.

  • 160.
    Pedersen, K
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. MOLEKYLÄRBIOLOGI.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    The bacterial toxin RelE displays codon specific cleavage of mRNAs in the ribosomal A-site2003In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 112, no 1, p. 131-140Article in journal (Refereed)
  • 161.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Barkefors, Irmeli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single molecule methods with applications in living cells2013In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 24, no 4, p. 737-744Article, review/survey (Refereed)
    Abstract [en]

    Our knowledge about dynamic processes in biological cells systems has been obtained roughly on two levels of detail; molecular level experiments with purified components in test tubes and system wide experiments with indirect readouts in living cells. However, with the development of single molecule methods for application in living cells, this partition has started to dissolve. It is now possible to perform detailed biophysical experiments at high temporal resolution and to directly observe processes at the level of molecules in living cells. In this review we present single molecule methods that can easily be implemented by readers interested to venture into this exciting and expanding field. We also review some recent studies where single molecule methods have been used successfully to answer biological questions as well as some of the most common pitfalls associated with these methods.

  • 162.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fritzsche, Joachim
    Mir, Kalim U.
    Modesti, Mauro
    Westerlund, Fredrik
    Tegenfeldt, Jonas O.
    Lipid-Based Passivation in Nanofluidics2012In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 12, no 5, p. 2260-2265Article in journal (Refereed)
    Abstract [en]

    Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. To facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.

  • 163.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Linden, Martin
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    A Bayesian Approach to Single Particle Tracking Analysis2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 177A-177AArticle in journal (Other academic)
  • 164.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Linden, Martin
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Extracting intracellular diffusive states and transition rates from single-molecule tracking data2013In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, no 3, p. 265-269Article in journal (Refereed)
    Abstract [en]

    We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.

  • 165. Pettersson, Mats
    et al.
    Besnier, Francois
    Siegel, Paul B.
    Carlborg, Örjan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. SLU.
    Replication and Explorations of High-Order Epistasis Using a Large Advanced Intercross Line Pedigree2011In: PLoS Genetics, ISSN 1553-7390, Vol. 7, no 7, p. e1002180-Article in journal (Refereed)
    Abstract [en]

    Dissection of the genetic architecture of complex traits persists as a major challenge in biology; despite considerable efforts, much remains unclear including the role and importance of genetic interactions. This study provides empirical evidence for a strong and persistent contribution of both second- and third-order epistatic interactions to long-term selection response for body weight in two divergently selected chicken lines. We earlier reported a network of interacting loci with large effects on body weight in an F(2) intercross between these high-and low-body weight lines. Here, most pair-wise interactions in the network are replicated in an independent eight-generation advanced intercross line (AIL). The original report showed an important contribution of capacitating epistasis to growth, meaning that the genotype at a hub in the network releases the effects of one or several peripheral loci. After fine-mapping of the loci in the AIL, we show that these interactions were persistent over time. The replication of five of six originally reported epistatic loci, as well as the capacitating epistasis, provides strong empirical evidence that the originally observed epistasis is of biological importance and is a contributor in the genetic architecture of this population. The stability of genetic interaction mechanisms over time indicates a non-transient role of epistasis on phenotypic change. Third-order epistasis was for the first time examined in this study and was shown to make an important contribution to growth, which suggests that the genetic architecture of growth is more complex than can be explained by two-locus interactions only. Our results illustrate the importance of designing studies that facilitate exploration of epistasis in populations for obtaining a comprehensive understanding of the genetics underlying a complex trait.

  • 166. Pilot, Malgorzata
    et al.
    Dabrowski, Michal J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Hayrapetyan, Vahram
    Yavruyan, Eduard G.
    Kopaliani, Natia
    Tsingarska, Elena
    Bujalska, Barbara
    Kaminski, Stanislaw
    Bogdanowicz, Wieslaw
    Genetic Variability of the Grey Wolf Canis lupus in the Caucasus in Comparison with Europe and the Middle East: Distinct or Intermediary Population?2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, p. e93828-Article in journal (Refereed)
    Abstract [en]

    Despite continuous historical distribution of the grey wolf (Canis lupus) throughout Eurasia, the species displays considerable morphological differentiation that resulted in delimitation of a number of subspecies. However, these morphological discontinuities are not always consistent with patterns of genetic differentiation. Here we assess genetic distinctiveness of grey wolves from the Caucasus (a region at the border between Europe and West Asia) that have been classified as a distinct subspecies C. l. cubanensis. We analysed their genetic variability based on mtDNA control region, microsatellite loci and genome-wide SNP genotypes (obtained for a subset of the samples), and found similar or higher levels of genetic diversity at all these types of loci as compared with other Eurasian populations. Although we found no evidence for a recent genetic bottleneck, genome-wide linkage disequilibrium patterns suggest a long-term demographic decline in the Caucasian population - a trend consistent with other Eurasian populations. Caucasian wolves share mtDNA haplotypes with both Eastern European and West Asian wolves, suggesting past or ongoing gene flow. Microsatellite data also suggest gene flow between the Caucasus and Eastern Europe. We found evidence for moderate admixture between the Caucasian wolves and domestic dogs, at a level comparable with other Eurasian populations. Taken together, our results show that Caucasian wolves are not genetically isolated from other Eurasian populations, share with them the same demographic trends, and are affected by similar conservation problems.

  • 167. Pimenta, A. C.
    et al.
    Dourado, Daniel F. A. R.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Martins, J. M.
    Melo, A.
    Dias Soeiro Cordeiro, M. N.
    Almeida, R. D.
    Morra, G.
    Moreira, I. S.
    Dynamic Structure of NGF and proNGF Complexed with p75NTR: Pro-Peptide Effect2014In: Journal of Chemical Information and Modeling, ISSN 1549-9596, Vol. 54, no 7, p. 2051-2067Article in journal (Refereed)
    Abstract [en]

    Crystallographic structures of NGF/p75NTR and proNGF/p75NTR were previously obtained in 2:1 and 2:2 stoichiometries, respectively. However, evidence shows that both stoichiometries can occur for mature neurotrophins and proneurotrophins. We used Molecular Dynamics (MD) simulations to examine the energetic and structural characteristics of these two complete systems as well as the uncomplexed forms of NGF and understand how these could translate in a new view of different biological outcomes. Here, we show that one chain at the 2:2 proNGF complex seems to be preferentially lost creating a 2:1 structure able to interact with sortilin. We also demonstrated that the structure of the neurotrophin dimers is not pre-established and suffers large structural modifications upon p75NTR binding. Moreover, our data suggests an elegant explanation for the dual role of NGF in neuronal cell death and survival, where different stoichiometries induce conformational changes that might be the basis for the different biological outcomes observed with the mature and proforms of neurotrophins.

  • 168.
    Poelstra, Jelmer W.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Vijay, Nagarjun
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bossu, Christen M.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Lantz, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ryll, Bettina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
    Mueller, I.
    Baglione, V.
    Unneberg, P.
    Wikelski, M.
    Grabherr, Manfred G.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wolf, Jochen B. W.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    The genomic landscape underlying phenotypic integrity in the face of gene flow in crows2014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 344, no 6190, p. 1410-1414Article in journal (Refereed)
    Abstract [en]

    The importance, extent, and mode of interspecific gene flow for the evolution of species has long been debated. Characterization of genomic differentiation in a classic example of hybridization between all-black carrion crows and gray-coated hooded crows identified genome-wide introgression extending far beyond the morphological hybrid zone. Gene expression divergence was concentrated in pigmentation genes expressed in gray versus black feather follicles. Only a small number of narrow genomic islands exhibited resistance to gene flow. One prominent genomic region (<2 megabases) harbored 81 of all 82 fixed differences (of 8.4 million single-nucleotide polymorphisms in total) linking genes involved in pigmentation and in visual perception-a genomic signal reflecting color-mediated prezygotic isolation. Thus, localized genomic selection can cause marked heterogeneity in introgression landscapes while maintaining phenotypic divergence.

  • 169. Prasad, B. Ram
    et al.
    Kamerlin, Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Florian, Jan
    Warshel, Arieh
    Prechemistry barriers and checkpoints do not contribute to fidelity and catalysis as long as they are not rate limiting2012In: Theoretical Chemistry accounts, ISSN 1432-881X, E-ISSN 1432-2234, Vol. 131, no 12, p. 1288-Article in journal (Refereed)
    Abstract [en]

    In the preceding article, "Perspective: Prechemistry conformational changes in DNA polymerase mechanisms" contributed by Schlick and coworkers as well as previous studies of these workers (Schlick et al. in Theor Chem Acc 131: 1287, 2012; Radhakrishnan and Schlick in J Am Chem Soc 127: 13245-13252, 2005; Radhakrishnan and Schlick in Biochem Biophys Res Commun 350: 521-529, 2006; Radhakrishnan et al. in Biochemistry 45: 15142-15156, 2006; Radhakrishnan and Schlick in Proc Natl Acad Sci USA 101: 5970-5975, 2004) have argued that the conformational changes preceding the chemical step contribute to DNA synthesis and to the fidelity of DNA polymerases. In one of our previous investigations (Ram Prasad and Warshel in Proteins 79:2900-2919, 2011), we argued and showed that as long as the free energy barriers associated with any of the prechemistry steps are not rate limiting, they could not contribute to the catalysis and then to the fidelity. Though all our arguments are based on exact and well-defined scientific logics, Schlick and coworkers seem to overlook some of the clear conditions in these arguments and in particular the requirement that the chemical step is rate limiting in their arguments that the prechemistry barriers contribute to the catalysis. In fact, as long as the prechemistry steps are not rate limiting, we have shown that the enzymes cannot carry the memory of the previous steps. We also address other potential misunderstandings about several key issues; First, we clarify that it is misleading to relate the prechemistry proposal to the clear fact that the substrate-induced conformational changes determine the final preorganization (the issue is the height of the barrier of the enzyme substrate system and not the trivial fact that the enzyme has to change its structure when the substrate binds). Second, we address the presumed role of dynamical effects in enzyme catalysis and the assumption that any observable should be explored in studies of biological function even if they are not relevant to the given effect. Third, we clarify that the fidelity cannot be explained or quantified by invoking the induced fit or conformational selection effects but by evaluating the free energy contributions to the rate-limiting steps from the structures of the corresponding systems (that of course can reflect the induce fit structural changes). Overall, we put a major emphasis on clarifying what is the prechemistry proposal and thus on trying to force the reader to focus on the only real controversy. We of course dismiss any implication that our studies cannot explore mutational effects as we actually pioneered such computational studies and we clarify that in studies of chemical rates, the focus must be placed on evaluating the chemical barriers, rather than on irrelevant factors, but that the calculations of the chemical barriers must consider all the factors that determine this barrier (including metal ions) and also examine if needed different problematic proposals such as dynamical effects, tunneling, and prechemistry.

  • 170.
    Prasad, Ram
    et al.
    University of Southern California, Department of Chemistry.
    Kamerlin, Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Plotnikov, Nikolay
    University of Southern California, Department of Chemistry.
    Warshel, Arieh
    University of Southern California, Department of Chemistry.
    Studying catalysis by QM/MM approaches should not be a black box process2012In: Thermodynamics and Catalysis, ISSN 2157-7544, Vol. 3, p. 4-Article in journal (Other academic)
  • 171. Pronk, Sander
    et al.
    Pall, Szilard
    Schulz, Roland
    Larsson, Per
    Bjelkmar, Par
    Apostolov, Rossen
    Shirts, Michael R.
    Smith, Jeremy C.
    Kasson, Peter M.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Hess, Berk
    Lindahl, Erik
    GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit2013In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 29, no 7, p. 845-854Article in journal (Refereed)
    Abstract [en]

    Motivation: Molecular simulation has historically been a low-throughput technique, but faster computers and increasing amounts of genomic and structural data are changing this by enabling large-scale automated simulation of, for instance, many conformers or mutants of biomolecules with or without a range of ligands. At the same time, advances in performance and scaling now make it possible to model complex biomolecular interaction and function in a manner directly testable by experiment. These applications share a need for fast and efficient software that can be deployed on massive scale in clusters, web servers, distributed computing or cloud resources. Results: Here, we present a range of new simulation algorithms and features developed during the past 4 years, leading up to the GROMACS 4.5 software package. The software now automatically handles wide classes of biomolecules, such as proteins, nucleic acids and lipids, and comes with all commonly used force fields for these molecules built-in. GROMACS supports several implicit solvent models, as well as new free-energy algorithms, and the software now uses multithreading for efficient parallelization even on low-end systems, including windows-based workstations. Together with hand-tuned assembly kernels and state-of-the-art parallelization, this provides extremely high performance and cost efficiency for high-throughput as well as massively parallel simulations.

  • 172. Przanowski, Piotr
    et al.
    Dabrowski, Michal
    Ellert-Miklaszewska, Aleksandra
    Kloss, Michal
    Mieczkowski, Jakub
    Kaza, Beata
    Ronowicz, Anna
    Hu, Feng
    Piotrowski, Arkadiusz
    Kettenmann, Helmut
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kaminska, Bozena
    The signal transducers Stat1 and Stat3 and their novel target Jmjd3 drive the expression of inflammatory genes in microglia2014In: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 92, no 3, p. 239-254Article in journal (Refereed)
    Abstract [en]

    Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of proinflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-. signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression.

  • 173.
    Repic, Matej
    et al.
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Vianello, Robert
    Quantum Organic Chemistry Group, Ruđer Bošković Institute, Zagreb, Croatia.
    Purg, Miha
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Lynn Shina Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mavri, Janez
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine2014In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 12, p. 3347-3355Article in journal (Refereed)
    Abstract [en]

    Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol(-1) compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol(-1), in excellent agreement with the experimental value of 16.5 kcal mol(-1). Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction.

  • 174. Rodriguez, Anna
    et al.
    Guerrero, Angel
    Gutierrez de Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Rodriguez, David
    Brea, Jose
    Loza, Maria I.
    Rosell, Gloria
    Bosch, M. Pilar
    New selective A(2A) agonists and A(3) antagonists for human adenosine receptors: synthesis, biological activity and molecular docking studies2015In: MedChemComm, ISSN 2040-2503, E-ISSN 2040-2511, Vol. 6, no 6, p. 1178-1185Article in journal (Refereed)
    Abstract [en]

    We report the synthesis and pharmacological characterization of a new series of adenosine derivatives on the four adenosine receptors (ARs). In radioligand binding assays, some of the compounds (1, 4, 6 and (R)-6) display a potent affinity for the A(2A)AR (K-i values < 10 nM) with high A(1)/A(2A) and A(2B)/A(2A) selectivity, moderate for the A(3)AR and low for the A(1)AR. The affinity of the epimeric mixture 6 was similar to that of the corresponding (R)-6 stereoisomer and 10-fold higher than that of the (S)-6 stereoisomer. The phenylethylamino group appears to play a key role on the activity, but introduction of groups of different sizes and electronegativity does not induce a substantial change in affinity for the A(2A)AR. In functional assays, most of the compounds produced similar amounts of cAMP compared to NECA, thus behaving as full A(2A)AR agonists. In addition, compounds 1, 2, 3, 5, (S)-6 and 9 resulted to be good antagonists for A(3)AR with K-B in the 6-14 nM range. Docking studies on the A(2A)AR showed a conserved binding mode consistent with previous A(2A)AR agonist-bound crystal structures, allowing for a rational interpretation of the SAR of this compound series.

  • 175. Rodriguez Carracedo, J.
    et al.
    de la Fuente Gonzalez, R. A.
    Varela Lara, I
    Brea Floriani, J.
    Rodriguez Diaz, D.
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Loza Garcia, M. , I
    Castro Perez, M.
    Residue HIS264 in Extracellular Loop 3 of Adenosine A(2A) Receptor is Critical for the Kinetics of Ligand Binding2014In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 115, no S2, p. 22-22, article id 56Article in journal (Other academic)
  • 176. Rogers, Kathleen E.
    et al.
    Keränen, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Durrant, Jacob D.
    Ratnam, Joseline
    Doak, Allison
    Arkin, Michelle R.
    McCammon, J. Andrew
    Novel Cruzain Inhibitors for the Treatment of Chagas' Disease2012In: Chemical Biology and Drug Design, ISSN 1747-0277, E-ISSN 1747-0285, Vol. 80, no 3, p. 398-405Article in journal (Refereed)
    Abstract [en]

    The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease, affects millions of individuals and continues to be an important global health concern. The poor efficacy and unfavorable side effects of current treatments necessitate novel therapeutics. Cruzain, the major cysteine protease of T.similar to cruzi, is one potential novel target. Recent advances in a class of vinyl sulfone inhibitors are encouraging; however, as most potential therapeutics fail in clinical trials and both disease progression and resistance call for combination therapy with several drugs, the identification of additional classes of inhibitory molecules is essential. Using an exhaustive virtual-screening and experimental validation approach, we identify several additional small-molecule cruzain inhibitors. Further optimization of these chemical scaffolds could lead to the development of novel drugs useful in the treatment of Chagas disease.

  • 177. Ryge, Marija Rakonjac
    et al.
    Tanabe, Michiharu
    Provost, Patrick
    Persson, Bengt
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Chen, Xinsheng
    Funk, Colin D.
    Rinaldo-Matthis, Agnes
    Hofmann, Bettina
    Steinhilber, Dieter
    Watanabe, Takashi
    Samuelsson, Bengt
    Radmark, Olof
    A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity2014In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 545, p. 179-185Article in journal (Refereed)
    Abstract [en]

    5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like beta-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the beta-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (v(init)) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix alpha 2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like beta-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX. (C) 2014 Elsevier Inc. All rights reserved.

  • 178. Saethre, Bjorn Steen
    et al.
    Hoffmann, Alex C.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Order Parameters and Algorithmic Approaches for Detection and Demarcation of Interfaces in Hydrate-Fluid and Ice-Fluid Systems2014In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 10, no 12, p. 5606-5615Article in journal (Refereed)
    Abstract [en]

    Some aspects of the use of order parameter fields in molecular dynamics simulations to delimit solid phases containing water, namely ice and hydrate, in both hydrophilic and hydrophobic fluids are examined; this includes the influences of rectangular meshes and of filtering on the quality of these parameters. Three order parameters are studied: the mass density, rho; an angular tetrahedrality measure, Sg (Chau and Hardwick, Mol. Phys. 1998, 93, 511); and the water-dimer dihedral angle, F-4 (Rodger et al. Fluid Phase Equilib. 1996, 116, 326). The parameters are studied to find their ability to distinguish between bulk phases, their consistency in different environments, their noise susceptibility, and their ability to demarcate the interface region. Spatial sampling and filtering are covered in detail, and some temporal features are illustrated by using autocorrelation maps. The parameters are employed to determine the position of interfaces as functions of time and, with the capillary wave fluctuation method (Hoyt et al. Phys. Rev. Lett. 2001, 86, 5530; Math. Comput. Simul. 2010, 80, 1382), to estimate solid-fluid interfacial stiffnesses, with partial success for the hydrophilic/hydrophobic-type interfaces.

  • 179. Saethre, Bjorn Steen
    et al.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Hoffmann, Alex C.
    Free Energy of Separation of Structure II Clathrate Hydrate in Water and a Light Oil2012In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 20, p. 5933-5940Article in journal (Refereed)
    Abstract [en]

    The adhesion forces and free energies of separation of structure II clathrate hydrates in vacuum and submerged in water and a model oil are investigated by molecular dynamics simulation. The water molecules are modeled by the TIP4P/ice model and the alkanes by the OPLS_AA force field. The results are compared with theory and earlier work. It is observed that the adhesive forces between the simulated surfaces have an effective range of no more than 1.5-2 nm. The hydrate hydrate interaction force is attractive in vacuum and oil, larger in vacuum. In water the interaction force is very slightly repulsive on average and much weaker than in the two other systems with a larger uncertainty. In all cases the interaction is largely entropically driven. The separation energies in vacuum and oil (octane) are stronger than predicted by theory, with free energies of approximately 4 and 0.7 aJ, respectively, likely due to lack of polarization effects. The hydrate hydrate interaction in water is too weak for quantitative comparisons to be made.

  • 180.
    Sanamrad, Arash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Biological Insights from Single-Particle Tracking in Living Cells2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time.

    Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers.

    Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

    List of papers
    1. Single-molecule investigations of the stringent response machinery in living bacterial cells
    Open this publication in new window or tab >>Single-molecule investigations of the stringent response machinery in living bacterial cells
    Show others...
    2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, p. E365-E373Article in journal (Refereed) Published
    Abstract [en]

    The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.

    Keywords
    cytosolic diffusion, single particle tracking, photoactivated localization microscopy, stroboscopic illumination
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-157244 (URN)10.1073/pnas.1102255108 (DOI)000293385700009 ()
    Available from: 2011-08-31 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
    2. Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
    Open this publication in new window or tab >>Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
    Show others...
    2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11413-11418Article in journal (Refereed) Published
    Abstract [en]

    Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

    Keywords
    nucleoid exclusion, transcription-translation coupling, antibiotics, single-molecule tracking, single-molecule imaging
    National Category
    Bioinformatics and Systems Biology
    Identifiers
    urn:nbn:se:uu:diva-229101 (URN)10.1073/pnas.1411558111 (DOI)000339807200043 ()25056965 (PubMedID)
    Available from: 2014-07-30 Created: 2014-07-30 Last updated: 2017-12-05Bibliographically approved
  • 181.
    Sanamrad, Arash
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundius, Ebba G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11413-11418Article in journal (Refereed)
    Abstract [en]

    Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

  • 182.
    Satpati, Priyadarshi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Energetic Tuning by tRNA Modifications Ensures Correct Decoding of Isoleucine and Methionine on the Ribosome2014In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 20, no 33, p. 10271-10275Article in journal (Refereed)
    Abstract [en]

    Chemical modifications of tRNAs are critical for accurate translation of the genetic code on the ribosome. The discrimination between isoleucine (AUA) and methionine (AUG) codons depends on such modifications of the wobble position in isoleucine tRNA anticodon loops, in all kingdoms of life. Bacteria and archaea employ functionally similar lysine- and agmatine-conjugated cytidine derivatives to ensure decoding fidelity, but the thermodynamics underlying codon discrimination remains unknown. Here, we report structure-based computer simulations that quantitatively reveal the energetics of this decoding strategy in archaea. The results further show that the agmatidine modification confers tRNA specificity primarily by desolvation of the incorrect codon in the non-cognate complex. Tautomerism is found to play no significant role in this decoding system as the usual amino form of the modified tRNA is by far the most stable.

  • 183.
    Satpati, Priyadarshi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sund, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Structure-Based Energetics of mRNA Decoding on the Ribosome2014In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, no 10, p. 1714-1722Article in journal (Refereed)
    Abstract [en]

    The origin of high fidelity in bacterial protein synthesis on the ribosome remains a fundamental unsolved problem despite available three-dimensional structures of different stages of the translation process. However, these structures open up the possibility of directly computing the energetics of tRNA selection that is required for an authentic understanding of fidelity in decoding. Here, we report extensive computer simulations that allow us to quantitatively calculate tRNA discrimination and uncover the energetics underlying accuracy in code translation. We show that the tRNA-mRNA interaction energetics varies drastically along the path from initial selection to peptide bond formation. While the selection process is obviously controlled by kinetics, the underlying thermodynamics explains the origin of the high degree of accuracy. The existence of both low- and high-selectivity states provides an efficient mechanism for initial selection and proofreading that does not require codon-dependent long-range structural signaling within the ribosome. It is instead the distinctly unequal population of the high-selectivity states for cognate and noncognate substrates that is the key discriminatory factor. The simulations reveal the essential roles played both by the 30S subunit conformational switch and by the common tRNA modification at position 37 in amplifying the accuracy.

  • 184.
    Satpati, Priyadarshi
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Why base tautomerization does not cause errors in mRNA decoding on the ribosome2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 20, p. 12876-12884Article in journal (Refereed)
    Abstract [en]

    The structure of the genetic code implies strict Watson-Crick base pairing in the first two codon positions, while the third position is known to be degenerate, thus allowing wobble base pairing. Recent crystal structures of near-cognate tRNAs accommodated into the ribosomal A-site, however, show canonical geometry even with first and second position mismatches. This immediately raises the question of whether these structures correspond to tautomerization of the base pairs. Further, if unusual tautomers are indeed trapped why do they not cause errors in decoding? Here, we use molecular dynamics free energy calculations of ribosomal complexes with cognate and near-cognate tRNAs to analyze the structures and energetics of G-U mismatches in the first two codon positions. We find that the enol tautomer of G is almost isoenergetic with the corresponding ketone in the first position, while it is actually more stable in the second position. Tautomerization of U, on the other hand is highly penalized. The presence of the unusual enol form of G thus explains the crystallographic observations. However, the calculations also show that this tautomer does not cause high codon reading error frequencies, as the resulting tRNA binding free energies are significantly higher than for the cognate complex.

  • 185. Serra-Vinardell, Jenny
    et al.
    Diaz, Lucia
    Guitierrez-de Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sanchez-Olle, Gessarni
    Bujons, Jordi
    Michelakakis, Helen
    Mavridou, Irene
    Aerts, Johannes M. F. G.
    Delgado, Antonio
    Grinberg, Daniel
    Vilageliu, Lluisa
    Casas, Josefina
    Selective chaperone effect of aminocyclitol derivatives on G202R and other mutant glucocerebrosidases causing Gaucher disease2014In: International Journal of Biochemistry and Cell Biology, ISSN 1357-2725, E-ISSN 1878-5875, Vol. 54, p. 245-254Article in journal (Refereed)
    Abstract [en]

    Gaucher disease is an autosomal recessive lysosomal disorder characterized by the accumulation of glucosylceramide as a result of a deficiency of the enzyme glucocerebrosidase. Several competitive glucocerebrosidase inhibitors are able to act as pharmacological chaperones for an efficient rescue of the mutated, misfolded forms of the enzyme. Along this line, we report in this work on the ability of several aminocyclitols to increase the residual glucocerebrosidase activity in patient fibroblasts with different genotypes. Some of the compounds were slightly active on fibroblasts bearing some mutations, including the highly prevalent N370S mutation. All compounds were highly active as enzyme activity enhancers on fibroblasts from Gaucher disease patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid omega-azidosphingosine, a reduction in the tagged glucosylceramide accumulation was also observed for selected aminocyclitols. Attempts to explain the activity impairment observed in glucocerebrosidase bearing the G202R mutation by comparative molecular dynamic studies on wild type and the G202R mutated proteins (free and isofagomine-bound, in both cases) were unsuccessful. Under the simulation conditions used, no clear effect of the G202R mutation neither over the global structure of the protein nor on the loops that constitute the glucocerebrosidase active site was observed. Since the G202R residue is located on the protein surface, altered protein-membrane or protein-protein interactions could account for the observed differences. In conclusion, we have tested novel compounds that have shown some chaperone effect on particular glucocerebrosidase mutant enzymes, supporting the enhancement therapy as an alternative approach for Gaucher disease. (C) 2014 Elsevier Ltd. All rights reserved.

  • 186.
    Shamsudin Khan, Yasmin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computational methods for calculating binding free energies of ligands in COX-12014Licentiate thesis, comprehensive summary (Other academic)
    List of papers
    1. Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors
    Open this publication in new window or tab >>Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors
    2014 (English)In: JOURNAL OF CHEMICAL INFORMATION AND MODELING, ISSN 1549-9596, Vol. 54, no 5, p. 1488-1499Article in journal (Refereed) Published
    Abstract [en]

    Cyclooxygenase-1 (COX-1) is one of the main targets of most pain-relieving pharmaceuticals. Although the enzyme is well characterized, it is known to be a difficult target for automated molecular docking and scoring. We collected from the literature a structurally diverse set of 45 nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors (coxibs) with a wide range of binding affinities for COX-1. The binding of this data set to a homology model of human COX-1 was analyzed with different combinations of molecular docking algorithms, scoring functions, and the linear interaction energy (LIE) method for estimating binding affinities. It is found that the computational protocols for estimation of binding affinities are extremely sensitive to the initial orientations of the ligands in the binding pocket. To overcome this limitation, we propose a systematic exploration of docking poses using the LIE calculations as a postscoring function. This scheme yields predictions in excellent agreement with experiment, with a mean unsigned error of 0.9 kcal/mol for binding free energies and structures of high quality. A significant improvement of the results is also seen when averaging over experimental data from several independent measurements.

    National Category
    Bioinformatics and Systems Biology
    Research subject
    Biology with specialization in Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-225268 (URN)10.1021/ci500151f (DOI)000336637400019 ()
    Available from: 2014-05-29 Created: 2014-05-29 Last updated: 2017-08-23Bibliographically approved
  • 187.
    Shamsudin Khan, Yasmin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Gutiérrez de Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Boukharta, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Toward an Optimal Docking and Free Energy Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors2014In: JOURNAL OF CHEMICAL INFORMATION AND MODELING, ISSN 1549-9596, Vol. 54, no 5, p. 1488-1499Article in journal (Refereed)
    Abstract [en]

    Cyclooxygenase-1 (COX-1) is one of the main targets of most pain-relieving pharmaceuticals. Although the enzyme is well characterized, it is known to be a difficult target for automated molecular docking and scoring. We collected from the literature a structurally diverse set of 45 nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors (coxibs) with a wide range of binding affinities for COX-1. The binding of this data set to a homology model of human COX-1 was analyzed with different combinations of molecular docking algorithms, scoring functions, and the linear interaction energy (LIE) method for estimating binding affinities. It is found that the computational protocols for estimation of binding affinities are extremely sensitive to the initial orientations of the ligands in the binding pocket. To overcome this limitation, we propose a systematic exploration of docking poses using the LIE calculations as a postscoring function. This scheme yields predictions in excellent agreement with experiment, with a mean unsigned error of 0.9 kcal/mol for binding free energies and structures of high quality. A significant improvement of the results is also seen when averaging over experimental data from several independent measurements.

  • 188.
    Shamsudin Khan, Yasmin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kazemi, Masoud
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-12015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 49, p. 7283-7291Article in journal (Refereed)
    Abstract [en]

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles.

  • 189. Shaw, Jeffrey J.
    et al.
    Trobro, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    He, Shan L.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Green, Rachel
    A Role for the 2 ' OH of Peptidyl-tRNA Substrate in Peptide Release on the Ribosome Revealed through RF-Mediated Rescue2012In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, no 8, p. 983-993Article in journal (Refereed)
    Abstract [en]

    The 2' OH of the peptidyl-tRNA substrate is thought to be important for catalysis of both peptide bond formation and peptide release in the ribosomal active site. The release reaction also specifically depends on a release factor protein (RF) to hydrolyze the ester linkage of the peptidyl-tRNA upon recognition of stop codons in the A site. Here, we demonstrate that certain amino acid substitutions (in particular those containing hydroxyl or thiol groups) in the conserved GGQ glutamine of release factor RF1 can rescue defects in the release reaction associated with peptidyl-tRNA substrates lacking a 2' OH. We explored this rescue effect through biochemical and computational approaches that support a model where the 2' OH of the P-site substrate is critical for orienting the nucleophile in a hydrogen-bonding network productive for catalysis.

  • 190.
    Shen, Xia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Novel Statistical Methods in Quantitative Genetics: Modeling Genetic Variance for Quantitative Trait Loci Mapping and Genomic Evaluation2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis develops and evaluates statistical methods for different types of genetic analyses, including quantitative trait loci (QTL) analysis, genome-wide association study (GWAS), and genomic evaluation. The main contribution of the thesis is to provide novel insights in modeling genetic variance, especially via random effects models.

    In variance component QTL analysis, a full likelihood model accounting for uncertainty in the identity-by-descent (IBD) matrix was developed. It was found to be able to correctly adjust the bias in genetic variance component estimation and gain power in QTL mapping in terms of precision. 

    Double hierarchical generalized linear models, and a non-iterative simplified version, were implemented and applied to fit data of an entire genome. These whole genome models were shown to have good performance in both QTL mapping and genomic prediction.

    A re-analysis of a publicly available GWAS data set identified significant loci in Arabidopsis that control phenotypic variance instead of mean, which validated the idea of variance-controlling genes. 

    The works in the thesis are accompanied by R packages available online, including a general statistical tool for fitting random effects models (hglm), an efficient generalized ridge regression for high-dimensional data (bigRR), a double-layer mixed model for genomic data analysis (iQTL), a stochastic IBD matrix calculator (MCIBD), a computational interface for QTL mapping (qtl.outbred), and a GWAS analysis tool for mapping variance-controlling loci (vGWAS).

    List of papers
    1. hglm: a package for fitting hierarchical generalized linear models
    Open this publication in new window or tab >>hglm: a package for fitting hierarchical generalized linear models
    2010 (English)In: The R Journal, ISSN 2073-4859, E-ISSN 2073-4859, Vol. 2, no 2, p. 20-28Article in journal (Refereed) Published
    Abstract [en]

    We present the hglm package for fitting hierarchical generalized linear models. It can be used for linear mixed models and generalized linear mixed models with random effects for a variety of links and a variety of distributions for both the outcomes and the random effects. Fixed effects can also be fitted in the dispersion part of the model.

    National Category
    Probability Theory and Statistics
    Identifiers
    urn:nbn:se:uu:diva-170083 (URN)000208590000004 ()
    Available from: 2012-03-07 Created: 2012-03-07 Last updated: 2017-12-07Bibliographically approved