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  • 1.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto, G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, p. 1526-1535Article in journal (Refereed)
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

  • 2. Bai, Shi
    et al.
    Jain, Mahendra K.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Contiguous binding of decylsulfate on the interface-binding surface of pancreatic phospholipase A22008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 9, p. 2899-2907Article in journal (Refereed)
    Abstract [en]

    Pig pancreatic IB phospholipase A(2) (PLA2) forms three distinguishable premicellar E-i(#) (i = 1, 2, and 3) complexes at successively higher decylsulfate concentrations. The Hill coefficient for E-1(#) is n(1) = 1.6, and n(2) and n(3) for E-2(#) and E-3(#) are about 8 each. Saturation-transfer difference nuclear magnetic resonance (NMR) and other complementary results with PLA2 show that decylsulfate molecules in E-2(#) and E-3(#) are contiguously and cooperatively clustered on the interface-binding surface or i-face that makes contact with the substrate interface. In these complexes, the saturation-transfer difference NMR signatures of H-1 in decylsulfate are different. The decylsulfate epitope for the successive E, complexes increasingly resembles the micellar complex formed by the binding of PLA2 to preformed micelles. Contiguous cooperative amphiphile binding is predominantly driven by the hydrophobic effect with a modest electrostatic shielding of the sulfate head group in contact with PLA2. The formation of the complexes is also associated with structural change in the enzyme. Calcium affinity of E-2(#) appears to be modestly lower than that of the free enzyme and Ell. Binding of decylsulfate to the i-face does not require the catalytic calcium required for the substrate binding to the active site and for the chemical step. These results show that E-i(#) complexes are useful to structurally characterize the cooperative sequential and contiguous binding of amphiphiles on the i-face. We suggest that the allosteric changes associated with the formation of discrete E-i(#) complexes are surrogates for the catalytic and allosteric states of the interface activated PLA2.

  • 3.
    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.

  • 4.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Jain, Mahendra K.
    Thermodynamic Reciprocity of the Inhibitor Binding to the Active Site and the Interface Binding Region of IB Phospholipase A22009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 14, p. 3209-3218Article in journal (Refereed)
    Abstract [en]

    Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E-i(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes ((E) over tilde (i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the (E) over tilde I-i complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the (E) over tilde (i) complexes with PCU are qualitatively similar to those of E-i(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E-2(#) complex or, equivalently, the shift in the E-1(#) to E-2(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.

  • 5.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Molecular Evolution.
    Jain, Mahendra
    Interfacial Enzyme Kinetics2002Book (Other academic)
    Abstract [en]

    This guide concentrates on the chemical and physical foundation of enzyme catalysis, the key area for the deeper understanding of biocatalytic processes. It also includes examples for proteins and nucleic acids, two central areas of biochemical and bio-organic research.

  • 6.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Apitz-Castro, Rafael J.
    Jain, Mahendra K.
    Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 7, p. 2080-2090Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E# with monodisperse diheptanoylphosphatidylcholine (DC7PC) that is distinguishable from the E* complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC7PC give an apparent dissociation constant of K2 = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E# are distinct from those of the E* complex. The aggregation and partitioning behaviors suggest that the acyl chains in E# but not in E* remain exposed to the aqueous phase. The free (E) and complexed (E# and E*) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC7PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis−Menten complex formed with monodisperse DC7PC is likely to be E#S or its aggregate rather than the classical monodisperse ES complex.

  • 7.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Chang, Cherry
    Koehler, Karl A.
    Jain, Mahendra K.
    Cooperative binding of monodisperse anionic amphiphiles to the i-Face: Phospholipase A2-paradigm for interfacial binding2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 25, p. 7999-8013Article in journal (Refereed)
    Abstract [en]

    Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A2 (PLA2) to form the premicellar complexes (Ei#) are characterized to discern the short-range specific interactions. Typically, Ei# complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (Ei#, i = 1, 2, 3). In the presence of calcium, the dissociation constant K1 for the E1# complex of PLA2 with decyl sulfate (CMC = 4500 μM) is 70 μM with a Hill coefficient n1 = 2.1 ± 0.2; K2 for E2# is 750 μM with n2 = 8 ± 1, and K3 for E3# is 4000 μM with an n3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of Ei#; (c) Ki and ni do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the Ei# complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in Ei#. The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of Ei#. We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and Ei# aggregates is discussed.

  • 8. Cajal, Yolanda
    et al.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Jain, Mahendra K.
    Origins of delays in monolayer kinetics: Phospholipase A2 paradigm2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 28, p. 9256-9264Article in journal (Refereed)
    Abstract [en]

    The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A2 (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow “activation” of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.

  • 9.
    Elf, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Paulsson, Johan
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mesoscopic kinetics and its applications in protein synthesis2005In: Systems Biology: Definitions and Perspectives / [ed] Lila Alberghina and H.V. Westerhoff, Heidelberg: Springer-Verlag GmbH , 2005, p. 95-118Chapter in book (Other academic)
    Abstract [en]

    Molecular biology emerged through unification of genetics and nucleic acid chemistry that took place with the discovery of the double helix (Watson and Crick 1953). Accordingly, molecular biology could be defined as the sum of all techniques used to perform genetic experiments by manipulating DNA. One consequence of the development of these techniques is large-scale sequencing of genomes from an ever increasing number of organisms. However, it became clear from this development that genetic information per se is not enough to grasp the most interesting functional and evolutionary aspects of cells and multi-cellular organisms. In fact, understanding how genotype leads to phenotype depends on concepts and techniques from areas that so far have been largely alien to molecular biological research, like physics, mathematics, and engineering. From the bits and pieces from these and other scientific fields new tools must be generated to make possible an understanding of the dynamic, adapting, and developing living systems that somehow take shape from the instructions given by their genomes. The growing total of these tools and their integration in experimental and theoretical approaches to understand complex biological processes in ways previously out of reach could be a way to define systems biology, in analogy with the above definition of molecular biology.

  • 10.
    Hammar, Petter
    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.
    Leroy, Prune
    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.
    Mahmutovic, Anel
    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.
    Marklund, Erik 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.
    Berg, Otto 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.
    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.
    The lac repressor displays facilitated diffusion in living cells2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 336, no 6088, p. 1595-1598Article in journal (Refereed)
    Abstract [en]

    Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO(1) operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.

  • 11.
    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.

  • 12. Jain, Mahendra Kumar
    et al.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Coupling of the i-face and the active site of phospholipase A2 for interfacial activation.2006In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 10, no 5, p. 473-479Article in journal (Refereed)
    Abstract [en]

    Interfacial enzymes bind to organized interfaces where they access their substrates. As an example of interfacial activation, phospholipase A2 has an observed rate of hydrolysis of the sn-2-acyl chain of phospholipids at bilayer and micellar interfaces that is more than 1,000 times larger than with monodisperse phospholipids. The major challenge for the study of interfacial enzymes is to correlate the elementary steps of the interfacial function of the enzyme with the structure of the enzyme at the interface. Having kinetically resolved the steps of the interfacial turnover cycle, here we outline our recent (mostly since 2000) approaches to address remaining issues of interfacial activation and also the protocols that are likely to provide insights into the distinguishing structural features of the interface-activated enzyme.

  • 13.
    Koskiniemi, Sanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sun, Song
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection-driven genome reduction in bacteria2012In: PLOS genetics, ISSN 1553-7404, Vol. 8, no 6, p. e1002787-Article in journal (Refereed)
    Abstract [en]

    Gene loss by deletion is a common evolutionary process in bacteria, as exemplified by bacteria with small genomes that have evolved from bacteria with larger genomes by reductive processes. The driving force(s) for genome reduction remains unclear, and here we examined the hypothesis that gene loss is selected because carriage of superfluous genes confers a fitness cost to the bacterium. In the bacterium Salmonella enterica, we measured deletion rates at 11 chromosomal positions and the fitness effects of several spontaneous deletions. Deletion rates varied over 200-fold between different regions with the replication terminus region showing the highest rates. Approximately 25% of the examined deletions caused an increase in fitness under one or several growth conditions, and after serial passage of wild-type bacteria in rich medium for 1,000 generations we observed fixation of deletions that substantially increased bacterial fitness when reconstructed in a non-evolved bacterium. These results suggest that selection could be a significant driver of gene loss and reductive genome evolution.

  • 14. Kurland, Charles G.
    et al.
    Canbäck, Björn
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    The origins of modem proteomes2007In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 89, no 12, p. 1454-1463Article in journal (Refereed)
    Abstract [en]

    Distributions of phylogenetically related protein domains (fold superfamilies), or FSFs, among the three Superkingdoms (trichotomy) are assessed. Very nearly 900 of the 1200 FSFs of the trichotomy are shared by two or three Superkingdoms. Parsimony analysis of FSF distributions suggests that the FSF complement of the last common ancestor to the trichotomy was more like that of modem eukaryotes than that of archaea and bacteria. Studies of length distributions among members of orthologous families of proteins present in all three Superkingdoms reveal that such lengths are significantly longer among eukaryotes than among bacteria and archaea. The data also reveal that proteins lengths of eukaryotes are more broadly distributed than they are within archaeal and bacterial members of the same orthologous families. Accordingly, selective pressure for a minimal size is significantly greater for orthologous protein lengths in archaea and bacteria than in eukaryotes. Alignments of orthologous proteins of archaea, bacteria and eukaryotes are characterized by greater sequence variation at their N-terminal and C-terminal domains, than in their central cores. Length variations tend to be localized in the terminal sequences; the conserved sequences of orthologous families are localized in a central core. These data are consistent with the interpretation that the genomes of the last common ancestor (LUCA) encoded a cohort of FSFs not very different from that of modem eukaryotes. Divergence of bacterial and archaeal genomes from that common ancestor may have been accompanied by more intensive reductive evolution of proteomes than that expressed in eukaryotes. Dollo's Law suggests that the evolution of novel FSFs is a very slow process, while laboratory experiments suggests that novel protein genesis from preexisting FSFs can be relatively rapid. Reassortment of FSFs to create novel proteins may have been mediated by genetic recombination before the advent of more efficient splicing mechanisms.

  • 15. Li, Gene-Wei
    et al.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioinformatics.
    Effects of macromolecular crowding and DNA looping on gene regulation kinetics.2009In: Nature Physics, ISSN 1745-2473, E-ISSN 1745-2481, Vol. 5, no 4, p. 294-297Article in journal (Refereed)
    Abstract [en]

    DNA-binding proteins control how genomes function. The theory of facilitated diffusion(1) explains how DNA-binding proteins can find targets apparently faster than the diffusion limit by using reduced dimensionality(2,3)-combining three-dimensional (3D) diffusion through cytoplasm with 1D sliding along DNA (refs 3-15). However, it does not include a description of macromolecular crowding on DNA as observed in living cells. Here, we show that such a physical constraint to sliding greatly reduces the search speed, in agreement with single-molecule measurements. Interestingly, the generalized theory also reveals significant insights into the design principles of biology. First, it places a hard constraint on the total number of DNA-binding proteins per cell. Remarkably, the number measured for Escherichia coli fits within the optimal range. Secondly, it defines a new role for DNA looping, a ubiquitous topological motif in genomes. DNA looping can speed up the search process by bypassing proteins that block the sliding track close to the target.

  • 16.
    Lind, Peter A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Umea Univ, Dept Mol Biol, S-90187 Umea, Sweden..
    Arvidsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Variation in Mutational Robustness between Different Proteins and the Predictability of Fitness Effects2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 2, p. 408-418Article in journal (Refereed)
    Abstract [en]

    Random mutations in genes from disparate protein classes may have different distributions of fitness effects (DFEs) depending on different structural, functional, and evolutionary constraints. We measured the fitness effects of 156 single mutations in the genes encoding AraC (transcription factor), AraD (enzyme), and AraE (transporter) used for bacterial growth on L-arabinose. Despite their different molecular functions these genes all had bimodal DFEs with most mutations either being neutral or strongly deleterious, providing a general expectation for the DFE. This contrasts with the unimodal DFEs previously obtained for ribosomal protein genes where most mutations were slightly deleterious. Based on theoretical considerations, we suggest that the 33-fold higher average mutational robustness of ribosomal proteins is due to stronger selection for reduced costs of translational and transcriptional errors. Whereas the large majority of synonymous mutations were deleterious for ribosomal proteins genes, no fitness effects could be detected for the AraCDE genes. Four mutations in AraC and AraE increased fitness, suggesting that slightly advantageous mutations make up a significant fraction of the DFE, but that they often escape detection due to the limited sensitivity of commonly used fitness assays. We show that the fitness effects of amino acid substitutions can be predicted based on evolutionary conservation, but those weakly deleterious mutations are less reliably detected. This suggests that large-effect mutations and the fraction of highly deleterious mutations can be computationally predicted, but that experiments are required to characterize the DFE close to neutrality, where many mutations ultimately fixed in a population will occur.

  • 17.
    Mahmutovic, Anel
    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.
    Berg, Otto 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.
    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.
    What matters for lac repressor search in vivo-sliding, hopping, intersegment transfer, crowding on DNA or recognition?2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, p. 3454-3464Article in journal (Refereed)
    Abstract [en]

    We have investigated which aspects of transcription factor DNA interactions are most important to account for the recent in vivo search time measurements for the dimeric lac repressor. We find the best agreement for a sliding model where non-specific binding to DNA is improbable at first contact and the sliding LacI protein binds at high probability when reaching the specific O-sym operator. We also find that the contribution of hopping to the overall search speed is negligible although physically unavoidable. The parameters that give the best fit reveal sliding distances, including hopping, close to what has been proposed in the past, i.e. similar to 40 bp, but with an unexpectedly high 1D diffusion constant on non-specific DNA sequences. Including a mechanism of inter-segment transfer between distant DNA segments does not bring down the 1D diffusion to the expected fraction of the in vitro value. This suggests a mechanism where transcription factors can slide less hindered in vivo than what is given by a simple viscosity scaling argument or that a modification of the model is needed. For example, the estimated diffusion rate constant would be consistent with the expectation if parts of the chromosome, away from the operator site, were inaccessible for searching.

  • 18.
    Mahmutovic, Anel
    et al.
    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.
    Berg, Otto G
    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.
    Elf, Johan
    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.
    What matters for lac repressor searchinvivo ––sliding, hopping, intersegment transfer, crowding on DNA or recognition?2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 7, p. 3454-3464Article in journal (Refereed)
    Abstract [en]

    We have investigated which aspects of transcription factor DNA interactions are most important to account for the recent in vivo search time measurements for the dimeric lac repressor. We find the best agreement for a sliding model where non-specific binding to DNA is improbable at first contact and the sliding LacI protein binds at high probability when reaching the specific Osym operator. We also find that the contribution of hopping to the overall search speed is negligible although physically unavoidable. The parameters that give the best fit reveal sliding distances, including hopping, close to what has been proposed in the past, i.e. ∼40 bp, but with an unexpectedly high 1D diffusion constant on non-specific DNA sequences. Including a mechanism of inter-segment transfer between distant DNA segments does not bring down the 1D diffusion to the expected fraction of the in vitro value. This suggests a mechanism where transcription factors can slide less hindered in vivo than what is given by a simple viscosity scaling argument or that a modification of the model is needed. For example, the estimated diffusion rate constant would be consistent with the expectation if parts of the chromosome, away from the operator site, were inaccessible for searching.

  • 19.
    Mahmutovic, Anel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Berg, Otto G.
    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.
    Lost in presumption: stochastic reactions in spatial models2012In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 9, no 12, p. 1163-1166Article in journal (Refereed)
    Abstract [en]

    Physical modeling is increasingly important for generating insights into intracellular processes. We describe situations in which combined spatial and stochastic aspects of chemical reactions are needed to capture the relevant dynamics of biochemical systems.

  • 20. Maisnier-Patin, Sophie
    et al.
    Roth, John
    Fredriksson, Åsa
    Nyström, Thomas
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Genomic buffering mitigates the effects of deleterious mutations in bacteria2005In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 37, no 12, p. 1376-1379Article in journal (Refereed)
  • 21.
    Marklund, Erik G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahmutovic, Anel
    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.
    Berg, Otto 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.
    Hammar, Petter
    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.
    van der Spoel, 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.
    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.
    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.
    Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 49, p. 19796-19801Article in journal (Refereed)
    Abstract [en]

    Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k(B)T for sliding and 12 kBT for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.

  • 22.
    Nilsson, Annika I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zorzet, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kanth, Anna
    Dahlström, Sabina
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 18, p. 6976-6981Article in journal (Refereed)
    Abstract [en]

    Deformylase inhibitors belong to a novel antibiotic class that targets peptide deformylase, a bacterial enzyme that removes the formyl group from N-terminal methionine in nascent polypeptides. Using the bacterium Salmonella enterica, we isolated mutants with resistance toward the peptide deformylase inhibitor actinonin. Resistance mutations were identified in two genes that are required for the formylation of methionyl (Met) initiator tRNA (tRNAi)(fMet): the fmt gene encoding the enzyme methionyl-tRNA formyltransferase and the folD gene encoding the bifunctional enzyme methylenetetrahydrofolate-dehydrogenase and -cyclohydrolase. In the absence of antibiotic, these resistance mutations conferred a fitness cost that was manifested as a reduced growth rate in laboratory medium and in mice. By serially passaging the low-fitness mutants in growth medium without antibiotic, the fitness costs could be partly ameliorated either by intragenic mutations in the fmt/folD genes or by extragenic compensatory mutations. Of the extragenically compensated fmt mutants, approximately one-third carried amplifications of the identical, tandemly repeated metZ and metW genes, encoding tRNAi. The increase in metZW gene copy number varied from 5- to 40-fold and was accompanied by a similar increase in tRNAi levels. The rise in tRNAi level compensated for the lack of methionyl-tRNA formyltransferase activity and allowed translation initiation to proceed with nonformylated methionyl tRNAi. Amplified units varied in size from 1.9 to 94 kbp. Suppression of deleterious mutations by gene amplification may be involved in the evolution of new gene functions.

  • 23. Nilsson, Annika
    et al.
    Kugelberg, Elisabeth
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Dan I.
    Karolinska Institute, Microbiology and Tumor Biology Center, S-171 77 Stockholm, Sweden.
    Experimental adaptation of Salmonella typhimurium to mice2004In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 168, p. 1119-1130Article in journal (Refereed)
    Abstract [en]

    Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization. By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment. Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected  into mice. After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11–0.58). Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10−6/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations. For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability. During adaptation there was no indication that a high mutation rate was counterselected. These data show that S. typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.                 

  • 24.
    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.

  • 25.
    Pettersson, Mats
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Dan
    Roth, John
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    The amplification model for adaptive mutation: simulations and analysis2005In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 169, no 2, p. 1105-1115Article in journal (Refereed)
    Abstract [en]

    It has been proposed that the lac revertants arising under selective conditions in the Cairns experiment do not arise by stress-induced mutagenesis of stationary phase cells as has been previously assumed. Instead, these revertants may arise within growing clones initiated by cells with a preexisting duplication of the weakly functional lac allele used in this experiment. It is proposed that spontaneous stepwise increases in lac copy number (amplification) allow a progressive improvement in growth.Reversion is made more likely primarily by the resultant increase in the number of mutational targets—more cells with more lac copies. The gene amplification model requires no stress-induced variation in the rate or target specificity of mutation and thus does not violate neo-Darwinian theory. However, it does require that a multistep process of amplification, reversion, and amplification segregation be completed within ≈ 20 generations of growth. This work examines the proposed amplification model from a theoretical point of view, formalizing it into a mathematical framework and using this to determine what would be required for the process to occur within the specified period. The analysis assumes no stress-induced change in mutation rate and describes only the growth improvement occurring during the process of amplification and subsequent elimination of excess mutant lac copies. The dynamics of the system are described using Monte Carlo simulations and numerical integration of the deterministic equations governing the system. The results imply that the amplification model can account for the behavior of the system using biologically reasonable parameter values and thus can, in principle, explain Cairnsian adaptive mutation.

  • 26.
    Pettersson, Mats E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Muller's ratchet in symbiont populations2007In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 130, no 2, p. 199-211Article in journal (Refereed)
    Abstract [en]

    Muller’s ratchet, the inevitable accumulation of deleterious mutations in asexual populations, has been proposed as a major factor in genome degradation of obligate symbiont organisms. Essentially, if left unchecked the ratchet will with certainty cause extinction due to the ever increasing mutational load. This paper examines the evolutionary fate of insect symbionts, using mathematical modelling to simulate the accumulation of deleterious mutations. We investigate the effects of a hierarchical two level population structure. Since each host contains its own subpopulation of symbionts, there will be a large number of small symbiont populations linked indirectly via selection on the host level. We show that although the separate subpopulations will accumulate deleterious mutations quickly, the symbiont population as a whole will be protected from extinction by selection acting on the hosts. As a consequence, the extent of genome degradation observed in present day symbionts is more likely to represent loss of functions that were (near-) neutral to the host, rather than a snap shot of a decline towards complete genetic collapse.

  • 27.
    Pettersson, Mats E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics.
    Kurland, Charles G.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Deletion rate evolution and its effect on genome size and coding density2009In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 26, no 6, p. 1421-1430Article in journal (Refereed)
    Abstract [en]

    Deletion rates are thought to be important factors in determining the genome size of organisms in nature. Although it is indisputable that deletions, and thus deletion rates, affect genome size, it is unclear how, or indeed if, genome size is regulated via the deletion rate. Here, we employ a mathematical model to determine the evolutionary fate of deletion rate mutants. Simulations are employed to explore the interactions between deletions, deletion rate mutants, and genome size. The results show that, in this model, the fate of deletion rate mutants   will depend on the fraction of essential genomic material, on the   frequency of sexual recombination, as well as on the population size of the organism. We find that there is no optimal deletion rate in any state. However, at one critical coding density, all changes in deletion rate are neutral and the rate may drift either up or down. As a consequence, the coding density of the genome is expected to fluctuate around this   critical density. Characteristic differences in the impact of deletion rate mutations on prokaryote and eukaryote genomes are described.

  • 28.
    Pettersson, Mats E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Sun, Song
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Evolution of new gene functions: simulation and analysis of the amplification model.2009In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 135, no 3, p. 309-324Article in journal (Refereed)
    Abstract [en]

    Creation of new genes and functions is a central feature of evolution. Duplication of existing genes has long been assumed to be the source of new genes, but the precise mechanism has remained unclear. One suggestion is that new genes are created via temporary amplifications, which simultaneously increase both the selective advantage of weak, pre-existing secondary functions and the target for optimizing mutations. This paper examines the amplification model by formalizing it into a mathematical framework. This framework is used to perform stochastic (Monte Carlo) simulations. In addition, experimental data from Salmonella typhimurium LT2 are used to support the modelling, by providing estimates for parameter values. The results show that amplification of tandem repeats is likely to contribute to creation of new genes in nature.

  • 29.
    Söderberg, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Mutational interference and the progression of Muller's ratchet when mutations have a broad range of deleterious effects2007In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 177, no 2, p. 971-986Article in journal (Refereed)
    Abstract [en]

    Deleterious mutations can accumulate in asexual haploid genomes through the process known as Muller's ratchet. This process has been described in the literature mostly for the case where all mutations are assumed to have the same effect on fitness. In the more realistic situation, deleterious mutations will affect fitness with a wide range of effects, from almost neutral to lethal. To elucidate the behavior of the ratchet in this more realistic case, simulations were carried out in a number of models, one where all mutations have the same effect on selection [one-dimensional (1D) model], one where the deleterious mutations can be divided into two groups with different selective effects [two-dimensional (2D) model], and finally one where the deleterious effects are distributed. The behavior of these models suggests that deleterious mutations can be classified into three different categories, such that the behavior of each can be described in a straightforward way. This makes it possible to predict the ratchet rate for an arbitrary distribution of fitness effects using the results for the well-studied 1D model with a single selection coefficient. The description was tested and shown to work well in simulations where selection coefficients are derived from an exponential distribution.

  • 30.
    Wistrand-Yuen, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Knopp, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution of high-level resistance during low-level antibiotic exposure2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, no 1, article id 1599Article in journal (Refereed)
    Abstract [en]

    It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

  • 31. Yu, Bao-Zhu
    et al.
    Apitz-Castro, Rafael J.
    Jain, Mahendra K.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A(2): Regulation of fat and cholesterol homeostasis2007In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1768, no 10, p. 2478-2490Article in journal (Refereed)
    Abstract [en]

    Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62–66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric kcat-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert EB complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.

  • 32. Yu, Bao-Zhu
    et al.
    Bai, Shi
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Jain, Mahendra K.
    Allosteric Effect of Amphiphile Binding to Phospholipase A22009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 14, p. 3219-3229Article in journal (Refereed)
    Abstract [en]

    In the preceding paper, we showed that the formation of the second premicellar complex of pig pancreatic IB phospholipase A2 (PLA2) can be considered a proxy for interface-activated substrate binding. Here we show that this conclusion is supported by results from premicellar;E-i(#) (i = 1, 2, or 3) complexes with a wide range of mutants of PLA2. Results also show a structural bass-for the correlated functional changes during the formation of E-2(#), and this is interpreted as an allosteric T (inactive) to R (active) transition. For example, the dissociation constant K-2(#) for decylsulfate bound to E-2(#) is lower at lower pH, at higher calcium concentrations, or with an inhibitor bound to the active site. Also, the lower limits of the K-2(#) values are comparable under these conditions. The pH-dependent increase in K-2(#) with a pK(a) of 6.5 is attributed to E71 which participates in the binding of the second calcium which in turn influences the enzyme binding to phosphatidylcholine interface. Most mutants exhibited kinetic and spectroscopic behavior that is comparable to that of native PLA2 and Delta PLA2 with a deleted 62-66 loop. However, the Delta Y52L substitution mutant cannot undergo the calcium-, pH-, or interface-dependent changes. We suggest that the Y52L substitution impairs the R to T transition and also hinders the approach of the Michaelis complex to the transition state. This allosteric change may be mediated by the structural motifs that connect the D48-D99 catalytic diad, the substrate-binding slot, and the residues of the i-face. Our interpretation is that the 57-72 loop and the H48DNCY52 segment of PLA2 are involved in transmitting the effect of the cooperative amphiphile binding to the i-face as a structural change in the active site.

  • 33. Yu, Bao-Zhu
    et al.
    Kaimal, Rajani
    Bai, Shi
    El Sayed, Khalid A.
    Tatulian, Suren A.
    Apitz, Rafael J.
    Jain, Mahendra K.
    Deng, Ruitang
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Effect of Guggulsterone and Cembranoids of Commiphora mukul on Pancreatic Phospholipase A(2): Role in Hypocholesterolemia2009In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 72, no 1, p. 24-28Article in journal (Refereed)
    Abstract [en]

    Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown to be specific modulators of two independent sites that are also modulated by bile salts (1-6) to control cholesterol absorption and catabolism. Guggulsterone (7) antagonized the chenodeoxycholic acid (3)-activated nuclear farnesoid X receptor (FXR), which regulates cholesterol metabolism in the liver. The cembranoids did not show a noticeable effect on FXR, but lowered the cholate (I)-activated rate of human pancreatic 113 phospholipase A2 (hPLA2), which controls gastrointestinal absorption of fat and cholesterol. Analysis of the data using a kinetic model has suggested an allosteric mechanism for the rate increase of hPLA2 by cholate and also for the rate-lowering effect by certain bile salts or cembranoids on the cholate-activated hPLA2 hydrolysis of phosphatidylcholine vesicles. The allosteric inhibition of PLA2 by certain bile salts and cembranoids showed some structural specificity. Biophysical studies also showed specific interaction of the bile salts with the interface-bound cholate-activated PLA2. Since cholesterol homeostasis in mammals is regulated by FXR in the liver for metabolism and by PLA2 in the intestine for absorption, modulation of PLA2 and FXR by bile acids and selected guggul components suggests novel possibilities for hypolipidemic and hypocholesterolemic therapies.

  • 34. Yu, Bao-Zhu
    et al.
    Polenova, Tatyana
    Jain, Mahendra Kumar
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover2005In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1712, no 2, p. 137-151Article in journal (Refereed)
    Abstract [en]

    During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121–131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC7PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (Ei#) of SMase with DC7PC. Biophysical studies indicate that in Ei# monodisperse DC7PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC7PC (Ei# with i = 1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction (X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC7PC will be monodisperse, Ei# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC7PC in solution is in a rapid exchange with that bound to Ei# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the Ei# complexes for the interpretation of the stationary phase reaction progress is discussed.

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