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8 Publications

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    Eddy/Rivas Lab
    05/30/08 | A probabilistic model of local sequence alignment that simplifies statistical significance estimation.
    Sean R. Eddy
    PLoS Computational Biology. 2008 May 30;4:e1000069. doi: 10.1371/journal.pcbi.1000069

    Sequence database searches require accurate estimation of the statistical significance of scores. Optimal local sequence alignment scores follow Gumbel distributions, but determining an important parameter of the distribution (lambda) requires time-consuming computational simulation. Moreover, optimal alignment scores are less powerful than probabilistic scores that integrate over alignment uncertainty ("Forward" scores), but the expected distribution of Forward scores remains unknown. Here, I conjecture that both expected score distributions have simple, predictable forms when full probabilistic modeling methods are used. For a probabilistic model of local sequence alignment, optimal alignment bit scores ("Viterbi" scores) are Gumbel-distributed with constant lambda = log 2, and the high scoring tail of Forward scores is exponential with the same constant lambda. Simulation studies support these conjectures over a wide range of profile/sequence comparisons, using 9,318 profile-hidden Markov models from the Pfam database. This enables efficient and accurate determination of expectation values (E-values) for both Viterbi and Forward scores for probabilistic local alignments.

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    05/30/08 | Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily.
    Brouns SJ, Barends TR, Worm P, Akerboom J, Turnbull AP, Salmon L, van der Oost J
    Journal of Molecular Biology. 2008 May 30;379:357-71. doi: 10.1016/j.jmb.2008.03.064

    The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 A resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded beta-sheet flanked by two alpha-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons alpha to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.

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    Tjian Lab
    05/06/08 | ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a.
    Gao X, Tate P, Hu P, Tjian R, Skarnes WC, Wang Z
    Proceedings of the National Academy of Sciences of the United States of America. 2008 May 6;105(18):6656-61. doi: 10.1073/pnas.1100640108

    ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectoderm-derived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.

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    05/01/08 | A cost-benefit analysis of neuronal morphology.
    Wen Q, Chklovskii DB
    Journal of Neurophysiology. 2008 May;99(5):2320-8. doi: 10.1371/journal.pcbi.1001066

    Over hundreds of millions of years, evolution has optimized brain design to maximize its functionality while minimizing costs associated with building and maintenance. This observation suggests that one can use optimization theory to rationalize various features of brain design. Here, we attempt to explain the dimensions and branching structure of dendritic arbors by minimizing dendritic cost for given potential synaptic connectivity. Assuming only that dendritic cost increases with total dendritic length and path length from synapses to soma, we find that branching, planar, and compact dendritic arbors, such as those belonging to Purkinje cells in the cerebellum, are optimal. The theory predicts that adjacent Purkinje dendritic arbors should spatially segregate. In addition, we propose two explicit cost function expressions, falsifiable by measuring dendritic caliber near bifurcations.

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    05/01/08 | Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin.
    Alfaro E, Sosa LD, Sanoguet Z, Pastrana-Ríos B, Schreiter ER
    Acta Crystallographica Section F Structural Biology and Crystallization Communications. 2008 May 1;64(Pt 5):402-4. doi: 10.1107/S1744309108009123

    Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 A resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 A, and are likely to belong to space group P2(1)2(1)2.

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    05/01/08 | Ethanol sensitivity and tolerance in long-term memory mutants of Drosophila melanogaster.
    Berger KH, Kong EC, Dubnau J, Tully T, Moore MS, Heberlein U
    Alcoholism, Clinical and Experimental Research. 2008 May;32(5):895-908. doi: 10.1111/j.1530-0277.2008.00659.x

    BACKGROUND: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants.

    METHODS: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure.

    RESULTS: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects.

    CONCLUSIONS: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

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    05/01/08 | Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.
    Shroff H, Galbraith CG, Galbraith JA, Betzig E
    Nature Methods. 2008 May;5(5):417-23. doi: 10.1038/nmeth.1202

    We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

    Commentary: The first example of true live cell and time lapse imaging by localization microscopy (as opposed to particle tracking), this paper uses the Nyquist criterion to establish a necessary condition for true spatial resolution based on the density of localized molecules – a condition often unmet in claims elsewhere in the superresolution literature.
    By any method, higher spatiotemporal resolution requires increasing light exposure at the specimen, making noninvasive imaging increasingly difficult. Here, simultaneous differential interference contrast imaging is used to establish that cells behave physiologically before, during, and after PALM imaging. Similar controls are lacking from many supposed “live cell” superresolution demonstrations.

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    05/01/08 | Super-resolution light microscopy goes live.
    Gustafsson MG
    Nature Methods. 2008 May;5(5):385-7. doi: 10.1038/nmeth0508-385

    Microscopic resolution far beyond the diffraction limit is possible by localizing single molecules individually. This approach has now been demonstrated on living cells.

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