The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels

<p>Abstract</p> <p>Background</p> <p>The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male <it>Drosophila </it>cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the <it>maleless </it>(<it>mle</it>) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted. To understand the molecular basis of Zn72D function, we identified proteins that interact with Zn72D.</p> <p>Results</p> <p>Among several proteins that associate with Zn72D, we found the DEAD box helicase Belle (Bel). Simultaneous knockdown of <it>Zn72D </it>and <it>bel </it>restored MSL complex localization to the X chromosome and dosage compensation. MLE protein was restored to 70% of wild-type levels, although the level of productively spliced <it>mle </it>transcript was still four-fold lower than in wild-type cells. The increase in production of MLE protein relative to the amount of correctly spliced <it>mle </it>mRNA could not be attributed to an alteration in MLE stability.</p> <p>Conclusion</p> <p>These data indicate that Zn72D and Bel work together to control <it>mle </it>splicing and protein levels. Thus Zn72D and Bel may be factors that coordinate splicing and translational regulation.</p

A Mathematical Programming Formulation for the Budding Yeast Cell Cycle

The budding yeast cell cycle can be modeled by a set of ordinary differential equations with 143 rate constant parameters. The quality of the model (and an associated vector of parameter settings) is measured by comparing simulation results to the experimental data derived from observing the cell cycles of over 100 selected mutated forms. Unfortunately, determining whether the simulated phenotype matches experimental data is difficult since the experimental data tend to be qualitative in nature (i.e., whether the mutation is viable, or which development phase it died in). Because of this, previous methods for automatically comparing simulation results to experimental data used a discontinuous penalty function, which limits the range of techniques available for automated estimation of the differential equation parameters. This paper presents a system of smooth inequality constraints that will be satisfied if and only if the model matches the experimental data. Results are presented for evaluating the mutants with the two most frequent phenotypes. This nonlinear inequality formulation is the first step toward solving a large-scale feasibility problem to determine the ordinary differential equation model parameters

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

AbstractSoft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Deterministic Parallel Global Parameter Estimation for a Model of the Budding Yeast Cell Cycle

Two parallel deterministic direct search algorithms are used to find improved parameters for a system of differential equations designed to simulate the cell cycle of budding yeast. Comparing the model simulation results to experimental data is difficult because most of the experimental data is qualitative rather than quantitative. An algorithm to convert simulation results to mutant phenotypes is presented. Vectors of parameters defining the differential equation model are rated by a discontinuous objective function. Parallel results on a 2200 processor supercomputer are presented for a global optimization algorithm, DIRECT, a local optimization algorithm, MADS, and a hybrid of the two

Detecting negative ions on board small satellites

Recent measurements near comets, planets, and their satellites have shown that heavy ions, energetic neutral atoms, molecular ions, and charged dust contain a wealth of information about the origin, evolution, and interaction of celestial bodies with their space environment. Using highly sensitive plasma instruments, positively charged heavy ions have been used to trace exospheric and surface composition of comets, planets, and satellites as well as the composition of interplanetary and interstellar dust. While positive ions dominate throughout the heliosphere, negative ions are also produced from surface interactions. In fact, laboratory experiments have shown that oxygen released from rocky surfaces is mostly negatively charged. Negative ions and negatively charged nanograins have been detected with plasma electron analyzers in several different environments (e.g., by Cassini and Rosetta), though more extensive studies have been challenging without instrumentation dedicated to negative ions. We discuss an adaptation of the Fast Imaging Plasma Spectrometer (FIPS) flown on MErcury Surface, Space ENvironment, GEochemistry and Ranging (MESSENGER) for the measurement of negatively charged particles. MESSENGER/FIPS successfully measured the plasma environment of Mercury from 2011 until 2015, when the mission ended, and has been used to map multiple ion species (H+ through Na+ and beyond) throughout Mercury’s space environment. Modifications to the existing instrument design fits within a 3U CubeSat volume and would provide a low mass, low power instrument, ideal for future CubeSat or distributed sensor missions seeking, for the first time, to characterize the contribution of negative particles in the heliospheric plasmas near the planets, moons, comets, and other sources.Key PointsSurface interactions with dust grains in the heliosphere and near the moon can produce anionsThe contribution of anions to the heliosphere and lunar environment is largely unknownAIPS is a small compact, yet capable anion sensor for use on small satellitesPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137433/1/jgra53416_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137433/2/jgra53416.pd

In-situ measurement of texture development rate in CaIrO₃ post-perovskite

The rate of crystallographic preferred orientation (CPO) development during deformation of post-perovskite is crucial in interpreting seismic anisotropy in the lowermost mantle but the stability field of MgSiO3 post-perovskite prevents high-strain deformation experiments being performed on it. Therefore, to constrain the rate of CPO development in post-perovskite, we deformed CaIrO3, a low-pressure analogue of MgSiO3 post-perovskite, in simple shear at 3.2GPa and 400○C to a shear strain (γ) of 0.81. From X-ray diffraction patterns acquired during deformation, we invert for CPO as a function of strain. By comparing the CPO that develops with visco-plastic self-consistent (VPSC) models we constrain the critical resolved shear stresses (CRSS) of the non-primary slip-systems in CaIrO3 to be of order 6 times stronger than the primary [100](010) slip system. This value is significantly less than has been assumed by previous studies and if applicable to MgSiO3 implies that seismic anisotropy in the D′ layer develops slower than has previously been assumed

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways

X Chromosomes Alternate between Two States prior to Random X-Inactivation

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells