DOCSLIB.ORG

Tandem Mass Spectrometry Identifies Many Mouse Brain O-Glcnacylated

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Tandem Mass Spectrometry Identifies Many Mouse Brain O-Glcnacylated Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets Joshua F. Alfaroa, Cheng-Xin Gongb, Matthew E. Monroea, Joshua T. Aldricha, Therese R. W. Claussa,SamuelO.Purvinea, Zihao Wangc, David G. Camp IIa, Jeffrey Shabanowitzd, Pamela Stanleye, Gerald W. Hartc, Donald F. Huntd, Feng Yanga,1, and Richard D. Smitha,1 aPacific Northwest National Laboratory, Richland, WA 99352; bDepartment of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314; cDepartment of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; dDepartment of Chemistry, University of Virginia, Charlottesville, VA 22904; and eDepartment of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461 Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 20, 2012 (received for review January 11, 2012) O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttransla- metric levels of O-GlcNAc modification at given sites on protein tional modification of Ser and Thr residues on cytosolic and nuclear substrates necessitate enrichment of O-GlcNAcylated proteins or proteins of higher eukaryotes catalyzed by O-GlcNAc transferase peptides before sequence analysis by MS (11). Additionally, iden- O tifying specific Ser and Thr residues that are O-GlcNAcylated is (OGT). -GlcNAc has recently been found on Notch1 extracellular do- fi O main catalyzed by EGF domain-specificOGT.AberrantO-GlcNAc mod- dif cult, because -GlcNAc is readily lost as an oxonium ion during ification of brain proteins has been linked to Alzheimer’s disease (AD). collision-induced dissociation (CID), a widely used fragmentation mode for peptide sequencing by MS (12). Alternative higher-energy However, understanding specific functions of O-GlcNAcylation in AD collisional dissociation (HCD) and electron transfer dissociation has been impeded by the difficulty in characterization of O-GlcNAc fi (ETD) MS methods have improved detection or have facilitated sites on proteins. In this study, we modi ed a chemical/enzymatic site-specific identification, but challenges remain (13). photochemical cleavage approach for enriching O-GlcNAcylated pep- Several methods to enrich O-GlcNAcylated proteins or pep- tides in samples containing ∼100 μg of tryptic peptides from mouse tides have led to the identification of a limited number of BIOCHEMISTRY cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins O-GlcNAcylation sites by MS. For example, lectin weak-affinity were identified. Of these, 168 were not previously known to be mod- chromatography (4, 10, 14) enabled identification of up to 142 ified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were O-GlcNAcylation sites in 62 proteins from mouse embryonic stem identified. Many of the modified residues are either known phosphor- cells (15). Immunoprecipitation, using O-GlcNAc-specific mono- ylation sites or located proximal to known phosphorylation sites. clonal antibodies (13, 16), identified 83 O-GlcNAcylated sites These findings support the proposed regulatory cross-talk between from a HEK293T cell extract (13). A recent metabolic labeling fi O-GlcNAcylation and phosphorylation. This study produced the most study that used alkyne-modi ed GlcNAc incorporated into OGT O substrates and Cu(I)-catalyzed [3 + 2] azide–alkyne cycloaddition comprehensive -GlcNAc proteome of mammalian brain tissue with – both protein identification and O-GlcNAc site assignment. Interest- (CuAAC) to a chemically cleavable biotin azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but ingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular yielded no information on sites of O-GlcNAc modification (17). In domains of five membrane proteins, expanding the evidence for ex- O O fi addition to limited coverage of -GlcNAcylation sites, a drawback tracellular -GlcNAcylation by the EGF domain-speci cOGT.Wealso to using these approaches is that they typically require milligram β α O fi report a GlcNAc- -1,3-Fuc- -1- -Thr modi cation on the EGF-like re- quantities of protein (4, 10, 13, 14, 16) or are limited to cultured peat of the versican core protein, a proposed substrate of Fringe β-1,3- cells (17), which makes them generally ill-suited for clinically de- N-acetylglucosaminyltransferases. rived tissue samples often available in small amounts. The chemical/enzymatic photochemical cleavage (CEPC) method chemical/enzymatic photochemical cleavage enrichment | glycosylation | (18, 19) improves upon the highly selective chemical/enzymatic mouse cerebral cortex approaches for O-GlcNAcylated proteins/peptides enrichment (20, 21) and increases analytical sensitivity by introducing a photo- O N O chemical cleavable-biotin probe that allows efficient release of single -linked -acetylglucosamine ( -GlcNAc) attached to fi Ser and Thr residues of cytosolic and nuclear proteins is a re- enriched peptides from the avidin af nity column. In this method A (Fig. 1A), O-GlcNAcylated peptides are first enzymatically labeled versible posttranslational modification (PTM) found in some bac- fi with azidogalactosamine (GalNAz). The free azido group in the teria, some protozoans, lamentous fungi, viruses, and all higher GalNAz is then conjugated to the alkyne group in a photocleavable eukaryotes. The enzymes that catalyze the dynamic cycling of biotin probe (PC-PEG-biotin-alkyne) through CuAAC. The bio- O-GlcNAc modification, O-GlcNAc transferase (OGT) and fi O tinylated peptides are enriched using avidin af nity chromatogra- -GlcNAc hexosaminidase (OGA), are more highly expressed in phy, and subsequently released through photochemical cleavage. the pancreas and brain than in other tissues (1, 2). In addition, O-GlcNAc-modified peptides enriched by this method are many proteins involved in neuronal communications, synaptic tagged with a basic aminomethyltriazolacetylgalactosamine (AMT- transmission, and synaptic plasticity are O-GlcNAcylated (3, 4), suggesting a role for this modification in brain function. O-GlcNAc cycling is highly sensitive to nutrients and stress and is regulated by Author contributions: J.F.A. and F.Y. designed research; J.F.A., T.R.W.C., and F.Y. per- nearly every metabolic pathway. Aberrant O-GlcNAc modification formed research; J.F.A., M.E.M., J.T.A., S.O.P., Z.W., F.Y., and R.D.S. contributed new has been linked to Alzheimer’s disease (AD) (5, 6) in which brain reagents/analytic tools; J.F.A., J.S., P.S., G.W.H., D.F.H., and F.Y. analyzed data; and J.F.A., glucose metabolism is impaired (7). The reduced O-GlcNAcylation C.-X.G., D.G.C., P.S., G.W.H., D.F.H., F.Y., and R.D.S. wrote the paper. in AD contributes to hyperphosphorylation of tau protein and The authors declare no conflict of interest. formation of the neurofibrillary tangles characteristic of AD and This article is a PNAS Direct Submission. related neurodegenerative disorders (8, 9). 1To whom correspondence may be addressed. E-mail: [email protected] or rds@pnnl. Current understanding of the function of O-GlcNAcylation in gov. fi neurodegeneration has been impeded by dif culties in identifying This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. this modification, even using MS (10, 11). First, the substoichio- 1073/pnas.1200425109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1200425109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Lysis A B WT mouse and 600 µg 20 mg cerebral trypsin desalted GalNAz transfer by corcal ssue digeson pepdes GalT1 Y289L, PNGase F 3xTg-AD mouse treatment, CIP 20 mg cerebral desalng corcal ssue 1/3 Saved Labeling with Bion-PEG-PC-Alkyne 1/3 1/3 SCX, Bion-avidin SCX, Bion-avidin Enrichment Enrichment Original Modified Wash Method Wash Method UV cleavage UV cleavage ½ sample ½ sample ½ sample CID-ETD HCD-CID/ETD HCD-CID/ETD WT 3xTg-AD WT 3xTg-AD WT 3xTg-AD *O-GlcNAc Sites: 358 152 160 135 182 139 O-GlcNAc Proteins: 249 133 126 104 144 122 Fig. 1. Overview of the CEPC method (A) and experimental work flow (B) used in this study for the identification of O-GlcNAc proteins and modification sites. (A) Steps modified in the enrichment strategy are depicted in dashed boxes. PNGase F and calf intestine phosphatase (CIP) are added to the reaction mixture to ensure selective and complete derivatization with GalNAz (18). During the CuAAC reaction, Cu(I) is stabilized with TBTA. (B) The original and modified wash methods for the biotin-avidin enrichment step were compared using peptides from one WT mouse and one 3xTg-AD mouse (female, 1 y old). The number of O-GlcNAc sites and proteins identified from LC-MS/MS analysis of individual samples is depicted at the bottom of the figure. GalNAc) that facilitates ETD identification and site localization of protein identifications by ∼16% (Fig. 1B) and reduced the large O-GlcNAc–modified peptides (10, 18, 19, 21). This approach en- amount of hydrophobic Tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] abled identification of 141 O-GlcNAcylation sites in 64 proteins amine (TBTA) that remained from the CuAAC reaction (Fig. S1). from <15 μg of spindle and midbody proteins that were enriched from HeLa cells (18, 19). However, to date, this method has not Evaluation of MS Fragmentation Methods for Identifying CEPC- been used for complex tissues or global proteomic analyses. In Enriched Tagged O-GlcNAcylated Peptides. In a parallel effort we addition, there remain challenges in reducing contaminants from evaluated the capability of individual MS/MS methods (HCD, CID, the CuAAC reaction, which are detrimental to liquid chromatog- and ETD) for identifying tagged O-GlcNAc (AMT-GalNAz- raphy (LC)-MS measurements. GlcNAc modified) in 100 μg WT mouse cerebrocortical peptides In the study reported herein, we modified the CEPC protocol enriched using CEPC. Of the three fragmentation methods, only and used it to enrich O-GlcNAcylated peptides from tissue samples ETD provided information regarding the location of the modifi- obtained from the brain cortex of one WT mouse and one 3xTg-AD cation sites.
Load more

Recommended publications
  • Cytogenomic SNP Microarray - Fetal ARUP Test Code 2002366 Maternal Contamination Study Fetal Spec Fetal Cells
    Patient Report |FINAL Client: Example Client ABC123 Patient: Patient, Example 123 Test Drive Salt Lake City, UT 84108 DOB 2/13/1987 UNITED STATES Gender: Female Patient Identifiers: 01234567890ABCD, 012345 Physician: Doctor, Example Visit Number (FIN): 01234567890ABCD Collection Date: 00/00/0000 00:00 Cytogenomic SNP Microarray - Fetal ARUP test code 2002366 Maternal Contamination Study Fetal Spec Fetal Cells Single fetal genotype present; no maternal cells present. Fetal and maternal samples were tested using STR markers to rule out maternal cell contamination. This result has been reviewed and approved by Maternal Specimen Yes Cytogenomic SNP Microarray - Fetal Abnormal * (Ref Interval: Normal) Test Performed: Cytogenomic SNP Microarray- Fetal (ARRAY FE) Specimen Type: Direct (uncultured) villi Indication for Testing: Patient with 46,XX,t(4;13)(p16.3;q12) (Quest: EN935475D) ----------------------------------------------------------------- ----- RESULT SUMMARY Abnormal Microarray Result (Male) Unbalanced Translocation Involving Chromosomes 4 and 13 Classification: Pathogenic 4p Terminal Deletion (Wolf-Hirschhorn syndrome) Copy number change: 4p16.3p16.2 loss Size: 5.1 Mb 13q Proximal Region Deletion Copy number change: 13q11q12.12 loss Size: 6.1 Mb ----------------------------------------------------------------- ----- RESULT DESCRIPTION This analysis showed a terminal deletion (1 copy present) involving chromosome 4 within 4p16.3p16.2 and a proximal interstitial deletion (1 copy present) involving chromosome 13 within 13q11q12.12. This
    [Show full text]
  • Network Medicine Approach for Analysis of Alzheimer's Disease Gene Expression Data
    International Journal of Molecular Sciences Article Network Medicine Approach for Analysis of Alzheimer’s Disease Gene Expression Data David Cohen y, Alexander Pilozzi y and Xudong Huang * Neurochemistry Laboratory, Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; [email protected] (D.C.); [email protected] (A.P.) * Correspondence: [email protected]; Tel./Fax: +1-617-724-9778 These authors contributed equally to this work. y Received: 15 November 2019; Accepted: 30 December 2019; Published: 3 January 2020 Abstract: Alzheimer’s disease (AD) is the most widespread diagnosed cause of dementia in the elderly. It is a progressive neurodegenerative disease that causes memory loss as well as other detrimental symptoms that are ultimately fatal. Due to the urgent nature of this disease, and the current lack of success in treatment and prevention, it is vital that different methods and approaches are applied to its study in order to better understand its underlying mechanisms. To this end, we have conducted network-based gene co-expression analysis on data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. By processing and filtering gene expression data taken from the blood samples of subjects with varying disease states and constructing networks based on that data to evaluate gene relationships, we have been able to learn about gene expression correlated with the disease, and we have identified several areas of potential research interest. Keywords: Alzheimer’s disease; network medicine; gene expression; neurodegeneration; neuroinflammation 1. Introduction Alzheimer’s disease (AD) is the most widespread diagnosed cause of dementia in the elderly [1].
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Mediator of DNA Damage Checkpoint 1 (MDC1) Is a Novel Estrogen Receptor Co-Regulator in Invasive 6 Lobular Carcinoma of the Breast 7 8 Evelyn K
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423142; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Running Title: MDC1 co-regulates ER in ILC 2 3 Research article 4 5 Mediator of DNA damage checkpoint 1 (MDC1) is a novel estrogen receptor co-regulator in invasive 6 lobular carcinoma of the breast 7 8 Evelyn K. Bordeaux1+, Joseph L. Sottnik1+, Sanjana Mehrotra1, Sarah E. Ferrara2, Andrew E. Goodspeed2,3, James 9 C. Costello2,3, Matthew J. Sikora1 10 11 +EKB and JLS contributed equally to this project. 12 13 Affiliations 14 1Dept. of Pathology, University of Colorado Anschutz Medical Campus 15 2Biostatistics and Bioinformatics Shared Resource, University of Colorado Comprehensive Cancer Center 16 3Dept. of Pharmacology, University of Colorado Anschutz Medical Campus 17 18 Corresponding author 19 Matthew J. Sikora, PhD.; Mail Stop 8104, Research Complex 1 South, Room 5117, 12801 E. 17th Ave.; Aurora, 20 CO 80045. Tel: (303)724-4301; Fax: (303)724-3712; email: [email protected]. Twitter: 21 @mjsikora 22 23 Authors' contributions 24 MJS conceived of the project. MJS, EKB, and JLS designed and performed experiments. JLS developed models 25 for the project. EKB, JLS, SM, and AEG contributed to data analysis and interpretation. SEF, AEG, and JCC 26 developed and performed informatics analyses. MJS wrote the draft manuscript; all authors read and revised the 27 manuscript and have read and approved of this version of the manuscript.
    [Show full text]
  • Prostate Cancer Prognostics Using Biomarkers Prostatakrebsprognostik Mittels Biomarkern Prognostic Du Cancer De La Prostate Au Moyen De Biomarqueurs
    (19) TZZ Z_T (11) EP 2 885 640 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 33/574 (2006.01) C12Q 1/68 (2018.01) 18.07.2018 Bulletin 2018/29 C40B 30/04 (2006.01) (21) Application number: 13829137.2 (86) International application number: PCT/US2013/055429 (22) Date of filing: 16.08.2013 (87) International publication number: WO 2014/028884 (20.02.2014 Gazette 2014/08) (54) PROSTATE CANCER PROGNOSTICS USING BIOMARKERS PROSTATAKREBSPROGNOSTIK MITTELS BIOMARKERN PROGNOSTIC DU CANCER DE LA PROSTATE AU MOYEN DE BIOMARQUEURS (84) Designated Contracting States: • GHADESSI, Mercedeh AL AT BE BG CH CY CZ DE DK EE ES FI FR GB New Westminster, British Columbia V3M 6E2 (CA) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • JENKINS, Robert, B. PL PT RO RS SE SI SK SM TR Rochester, Minnesota 55902 (US) • VERGARA CORREA, Ismael A. (30) Priority: 16.08.2012 US 201261684066 P Bundoora, Victoria 3083 (AU) 13.02.2013 US 201361764365 P 14.03.2013 US 201361783124 P (74) Representative: Cornish, Kristina Victoria Joy et al Kilburn & Strode LLP (43) Date of publication of application: Lacon London 24.06.2015 Bulletin 2015/26 84 Theobalds Road London WC1X 8NL (GB) (73) Proprietors: • Genomedx Biosciences, Inc. (56) References cited: Vancouver BC V6B 2W9 (CA) WO-A1-2009/143603 WO-A1-2013/090620 • MAYO FOUNDATION FOR MEDICAL WO-A2-2006/091776 WO-A2-2006/110264 EDUCATION AND RESEARCH WO-A2-2007/056049 US-A1- 2006 134 663 Rochester, MN 55905 (US) US-A1- 2007 037 165 US-A1- 2007 065 827 US-A1-
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Building the Vertebrate Codex Using the Gene Breaking Protein Trap Library
    Genetics, Development and Cell Biology Publications Genetics, Development and Cell Biology 2020 Building the vertebrate codex using the gene breaking protein trap library Noriko Ichino Mayo Clinic Gaurav K. Varshney National Institutes of Health Ying Wang Iowa State University Hsin-kai Liao Iowa State University Maura McGrail Iowa State University, [email protected] See next page for additional authors Follow this and additional works at: https://lib.dr.iastate.edu/gdcb_las_pubs Part of the Molecular Genetics Commons, and the Research Methods in Life Sciences Commons The complete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ gdcb_las_pubs/261. For information on how to cite this item, please visit http://lib.dr.iastate.edu/howtocite.html. This Article is brought to you for free and open access by the Genetics, Development and Cell Biology at Iowa State University Digital Repository. It has been accepted for inclusion in Genetics, Development and Cell Biology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Building the vertebrate codex using the gene breaking protein trap library Abstract One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene- break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown.
    [Show full text]
  • WO 2014/135655 Al 12 September 2014 (12.09.2014) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/135655 Al 12 September 2014 (12.09.2014) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/EP2014/054384 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 6 March 2014 (06.03.2014) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (30) Priority Data: ZW. 13305253.0 6 March 2013 (06.03.2013) EP (84) Designated States (unless otherwise indicated, for every (71) Applicants: INSTITUT CURIE [FR/FR]; 26 rue d'Ulm, kind of regional protection available): ARIPO (BW, GH, F-75248 Paris cedex 05 (FR). CENTRE NATIONAL DE GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, LA RECHERCHE SCIENTIFIQUE [FR/FR]; 3 rue UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Michel Ange, F-75016 Paris (FR).
    [Show full text]
  • Supp Material.Pdf
    Simon et al. Supplementary information: Table of contents p.1 Supplementary material and methods p.2-4 • PoIy(I)-poly(C) Treatment • Flow Cytometry and Immunohistochemistry • Western Blotting • Quantitative RT-PCR • Fluorescence In Situ Hybridization • RNA-Seq • Exome capture • Sequencing Supplementary Figures and Tables Suppl. items Description pages Figure 1 Inactivation of Ezh2 affects normal thymocyte development 5 Figure 2 Ezh2 mouse leukemias express cell surface T cell receptor 6 Figure 3 Expression of EZH2 and Hox genes in T-ALL 7 Figure 4 Additional mutation et deletion of chromatin modifiers in T-ALL 8 Figure 5 PRC2 expression and activity in human lymphoproliferative disease 9 Figure 6 PRC2 regulatory network (String analysis) 10 Table 1 Primers and probes for detection of PRC2 genes 11 Table 2 Patient and T-ALL characteristics 12 Table 3 Statistics of RNA and DNA sequencing 13 Table 4 Mutations found in human T-ALLs (see Fig. 3D and Suppl. Fig. 4) 14 Table 5 SNP populations in analyzed human T-ALL samples 15 Table 6 List of altered genes in T-ALL for DAVID analysis 20 Table 7 List of David functional clusters 31 Table 8 List of acquired SNP tested in normal non leukemic DNA 32 1 Simon et al. Supplementary Material and Methods PoIy(I)-poly(C) Treatment. pIpC (GE Healthcare Lifesciences) was dissolved in endotoxin-free D-PBS (Gibco) at a concentration of 2 mg/ml. Mice received four consecutive injections of 150 μg pIpC every other day. The day of the last pIpC injection was designated as day 0 of experiment.
    [Show full text]
  • Quantitative Live Cell Imaging Reveals Influenza Virus Manipulation Of
    ARTICLE https://doi.org/10.1038/s41467-019-13838-3 OPEN Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association Amar R. Bhagwat 1, Valerie Le Sage1, Eric Nturibi1, Katarzyna Kulej2, Jennifer Jones 1, Min Guo3, Eui Tae Kim 2, Benjamin A. Garcia4,5, Matthew D. Weitzman2,5,6, Hari Shroff3 & Seema S. Lakdawala 1,7* fl 1234567890():,; Assembly of infectious in uenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection. 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, PA 15219, USA. 2 The Children’s Hospital of Philadelphia Research Institute, 3501 Civic Center Dr., Philadelphia, PA 19104, USA. 3 Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 13 South Drive, Building 13, Bethesda, MD 20892, USA.
    [Show full text]
  • Bioinformatics Approaches to Identify Pain Mediators, Novel Lncrnas and Distinct Modalities of Neuropathic Pain
    Bioinformatics approaches to identify pain mediators, novel LncRNAs and distinct modalities of neuropathic pain by Georgios Baskozos A thesis submitted to University College London for the degree of Doctor of Philosophy Institute of Structural and Molecular Biology University College London September 2016 1 Declaration I, Georgios Baskozos, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ……………………………………… Georgios Baskozos 29 September 2016 2 Abstract This thesis presents a number of studies in the general subject of bioinformatics and functional genomics. The studies were made in collaboration with experimental scientists of the London Pain Consortium (LPC), an initiative that has promoted collaborations between experimental and computational scientists to further understanding of pain. The studies are mainly concerned with the molecular biology of pain and deal with data gathered from high throughput technologies aiming to assess the transcriptional changes involved in well induced pain states, both from animal models of pain and human patients. We have analysed next generation sequencing data (NGS data) in order to assess the transcriptional changes in rodent’s dorsal root ganglions under well induced pain states. We have also developed a customised computational pipeline to analyse RNA- sequencing data in order to identify novel Long non-coding RNAs (LncRNAs), which may function as mediators of neuropathic pain. Our analyses detected hundreds of novel LncRNAs significantly dysregulated between sham-operated animals and animal models of pain. In addition, in order to gain valuable insights into neuropathic pain, including both its molecular signature, somatosensory profiles and clusters of individuals related to pain severity, we analysed clinical data together with data obtained from quality of life pain-questionnaires.
    [Show full text]