DOCSLIB.ORG

Eleven Daughters of NANOG

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Eleven Daughters of NANOG Genomics 84 (2004) 229–238 www.elsevier.com/locate/ygeno Eleven daughters of NANOG$ H. Anne F. Booth and Peter W.H. Holland* Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK Received 4 December 2003; accepted 25 February 2004 Available online 15 April 2004 Abstract Nanog is a recently discovered ANTP class homeobox gene. Mouse Nanog is expressed in the inner cell mass and in embryonic stem cells and has roles in self-renewal and maintenance of pluripotency. Here we describe the location, genomic organization, and relative ages of all human NANOG pseudogenes, comprising ten processed pseudogenes and one tandem duplicate. These are compared to the original, intact human NANOG gene. Eleven is an unusually high number of pseudogenes for a homeobox gene and must reflect expression in the human germ line. A pseudogene orthologous to NANOGP4 was found in chimpanzee and an expressed pseudogene in macaque. Examining pseudogenes of differing ages gives insight into pseudogene decay, which involves an excess of deletion mutations over insertions. The mouse genome has two processed pseudogenes, which are not clear orthologues of the primate pseudogenes. D 2004 Elsevier Inc. All rights reserved. Keywords: Homeobox; Pseudogene; Molecular evolution; Enk Homeobox genes are characterized by possession of a Mitsui and colleagues [3] identified a single human recognizable 60- or 63-amino-acid motif within the orthologue of the mouse Nanog gene, mapping to encoded protein. The homeobox gene superfamily is 12p13.31. extremely diverse, particularly within animal genomes, As of February 2004, the Mouse Genome Informatics and can be subdivided into several major classes, each web site and National Center for Biotechnology Information containing numerous gene families. Among the best (NCBI) LocusLink list Nanog as the approved symbol for known are the Hox, En, Dlx, Evx, NK, and Msx gene the mouse gene (LocusID 71950), with Enk as an alternative families within the ANTP class, and the Pax, Gsc, and symbol. For the human gene, NCBI LocusLink lists Otx gene families within the PRD class. Recently, Wang NANOG as the gene symbol (LocusID 79923), with and colleagues [1] described the cloning of a novel FLJ12581 as an alternative symbol. Because the symbol member of the ANTP class in mouse, which they named Enk might cause confusion with the unrelated enkephalin Enk (early embryo specific expression NK family gene), gene, we refer to the mouse and human orthologues as referring to expression in inner cell mass cells of the Nanog and NANOG, respectively. blastocyst and sequence similarity to several NK-related Here we report the existence of ten processed homeobox genes. The same mouse gene was also cloned NANOG pseudogenes in the complete human genome, by Chambers and colleagues [2] and by Mitsui and plus one duplication pseudogene, and present full colleagues [3]; these groups showed that the encoded descriptions of their locations, organization, and relative protein plays key roles in self-renewal and maintenance ages. We also describe one pseudogene each from of pluripotency in inner cell mass and embryonic stem chimpanzee and macaque (more are expected) and two cells. Reflecting these properties, these authors designated in the complete mouse genome. We comment on the the gene Nanog, from Tı´r nan O´ g, the mythical Celtic process of pseudogene decay and propose explanations land of the ever young. Chambers and colleagues [2] and for the abundance of Nanog pseudogenes and for the particular abundance of primate pseudogenes. First, how- $ Supplementary data for this article may be found on ScienceDirect. ever, we clarify the sequence and organization of the * Corresponding author. Fax: +44-01865-271184. functional human NANOG gene, to allow comparison to E-mail address: [email protected] (P.W.H. Holland). the pseudogenes. 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.02.014 230 H.A.F. Booth, P.W.H. Holland / Genomics 84 (2004) 229–238 Results and discussion an unlinked gene (CDT1), probably artifactually. NANOG ESTs are primarily from human NT2 teratocarcinoma cells, Structure of human NANOG germ cell tumors, testis tumors, colon tumor, and adult marrow. The remaining 5 ESTs and the cDNA derive from We subjected the NCBI human genome assembly to pseudogenes and are discussed later. The NCBI contig tBLASTn searches using homeodomain sequences encoded annotation describes four exons; our analyses confirm this, by three human ANTP class genes: EN2, HOXA1, and with all splice sites obeying the GT–AG donor–acceptor NKX1.1. Among the retrieved sequences, three were home- rule [4]. The homeobox is located in exons 2 and 3 of the odomains from unnamed homeobox genes. A second four-exon gene, being interrupted between homeodomain tBLASTn search of the human genome using one of these residues 44 and 45. When an intron occurs within a unidentified homeodomains revealed several other highly homeobox, the most frequent site is between codons 44 similar sequences. One of these was an unnamed homeobox and 45 [5]. There are numerous other examples of this gene mapping to chromosome 12 at 12p13.31 (chromosom- intron location in the ANTP class, including several (but al position 7.84 Mb) and located on a genomic contig with not all) members of the Evx, Dlx, Lbx, Not, Gsx, and Hox RefSeq Accession No. NT_009714. This gene is clearly the gene families (e.g., Drosophila lab, pb, Abd-B). The human NANOG gene. Supporting evidence includes two human NANOG gene structure is shown diagrammatically 2.1kb cDNAs with GenBank Accession Nos. AK022643 in Fig. 1A. (RefSeq mRNA NM_024865) deposited by T. Isogai The genomic and mRNA sequences of NANOG are not (Chiba, Japan; unpublished) and AB093576 deposited by identical at the DNA level. In addition to several synony- S. Yamanaka (Nara, Japan [3]) and noted to be the human mous substitutions, there is one nonsynonymous difference orthologue of mouse Nanog. in exon 2, causing a difference at amino acid position 82: To confirm exon–intron organization, we searched lysine in the genomic contig and asparagine in the deposited GenBank and the NCBI human EST database and exam- full cDNA sequences. Lys and Asn are not biochemically ined the UniGene cluster for NANOG (Hs.326290). This similar amino acids, so this difference was unexpected. To yielded 33 EST sequences and a cDNA, in addition to the test if this is a naturally occurring polymorphism or a two cDNAs mentioned above. Manual comparisons indi- sequencing error, we used PCR to amplify human NANOG cated that 28 ESTs derive from the functional NANOG exon 2 from the genomic DNA of two individuals. We gene; of these, 20 match the known cDNA closely, 2 have found that one individual encoded Lys at position 82; the intron sequences (and therefore derive from unspliced other encoded Asn. This confirms there is naturally occur- message), and 4 (from the same library) are hybrids with ring variation in the NANOG protein sequence. Other non- Fig. 1. (A) Human NANOG gene structure. The four exons (labeled 1–4) are represented by horizontal bars; the 5V and 3V UTRs are white and the protein coding region is black with the homeobox colored blue. The three introns are represented by pink lines. The length in nucleotides is written underneath each of the exons and introns (with exons 1 and 4 being split into coding and noncoding/UTR regions). Note that the 3V UTR is 967 nucleotides in the version of genomic sequence deposited, but 983 nucleotides in the deposited cDNA sequences. The start (ATG) and stop (TGA) codons are labeled and represented by a green and a red vertical line, respectively. (B) Nine processed pseudogenes compared to human NANOG protein. One processed pseudogene, NANOGP11,is not shown, as it does not include sequence derived from the NANOG open reading frame. Chromosomal positions, RefSeq Accession Nos., and GenBank Accession Nos. are shown. The NANOG protein is represented by a black horizontal bar with the homeodomain colored blue. The start codon is represented by a green vertical line and the stop codon by a red vertical line. Intron positions are shown by pink triangles. For each pseudogene the black horizontal bar represents sequence similarity to the NANOG open reading frame, which can move between pseudogene reading frames (rf1, rf2, rf3) as a result of insertions and deletions. Insertions are represented by turquoise vertical lines and deletions by the absence of sequence similarity to NANOG. Below each insertion is a plus sign and below each deletion is a minus sign, followed by the number of nucleotide bases inserted/deleted at that point. Substitution mutations are not shown, except where these introduce stop codons into the reading frame homologous to NANOG. Red vertical lines indicate stop codons; these are shown when they occur in the reading frame homologous to NANOG or when they are the first stop codon encountered in rf1. In four of the pseudogenes (NANOGP2, P5, P6, P9) insertions or deletions have caused reading-frame shifts before a stop codon was encountered in rf1. In these cases, the gray dashed lines indicate continuation of rf1 until the first stop codon is encountered. Fig. 2. (A) Genes affected by tandem duplication of human NANOG. The tandem duplicates NANOG and NANOGP1 are represented by black boxes and the tandem duplicates SLC2A14 and SLC2A3 are represented by red boxes. The NANOGP1 box represents the region of homology to NANOG, not the full NANOGP1 transcript. NANOG and NANOGP1 are on the same DNA strand, while SLC2A14 and SLC2A3 are on the opposite strand (indicated by arrows underneath the genes). The intergenic distances are shown in kilobases and a scale bar of human chromosome 12 in megabases is drawn underneath.
Load more

Recommended publications
  • Nuclear Titin Interacts with A- and B-Type Lamins in Vitro and in Vivo
    Research Article 239 Nuclear Titin interacts with A- and B-type lamins in vitro and in vivo Michael S. Zastrow1,*, Denise B. Flaherty2‡, Guy M. Benian2 and Katherine L. Wilson1,§ 1Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA 2Department of Pathology, Emory University, Whitehead Biomedical Research Building, Atlanta, GA 30332, USA *Present address: Department of Developmental Biology, Stanford University School of Medicine, Beckman Center, B300, 279 Campus Drive, Stanford, CA 94305, USA ‡Department of Biology, Eckerd College, SHB 105, 4200 54th Ave, St Petersburg, FL 33711, USA §Author for correspondence (e-mail: [email protected]) Accepted 4 October 2005 Journal of Cell Science 119, 239-249 Published by The Company of Biologists 2006 doi:10.1242/jcs.02728 Summary Lamins form structural filaments in the nucleus. Mutations lamin-downregulated [lmn-1(RNAi)] embryos, Ce-titin was in A-type lamins cause muscular dystrophy, undetectable at the nuclear envelope suggesting its cardiomyopathy and other diseases, including progeroid localization or stability requires Ce-lamin. In human cells syndromes. To identify new binding partners for lamin A, (HeLa), antibodies against the titin-specific domain M-is6 we carried out a two-hybrid screen with a human skeletal- gave both diffuse and punctate intranuclear staining by muscle cDNA library, using the Ig-fold domain of lamin A indirect immunofluorescence, and recognized at least three as bait. The C-terminal region of titin was recovered twice. bands larger than 1 MDa in immunoblots of isolated HeLa Previous investigators showed that nuclear isoforms of titin nuclei.
    [Show full text]
  • Nesprins: from the Nuclear Envelope and Beyond
    expert reviews http://www.expertreviews.org/ in molecular medicine Nesprins: from the nuclear envelope and beyond Dipen Rajgor and Catherine M. Shanahan* Nuclear envelope spectrin-repeat proteins (Nesprins), are a novel family of nuclear and cytoskeletal proteins with rapidly expanding roles as intracellular scaffolds and linkers. Originally described as proteins that localise to the nuclear envelope (NE) and establish nuclear-cytoskeletal connections, nesprins have now been found to comprise a diverse spectrum of tissue specific isoforms that localise to multiple sub-cellular compartments. Here, we describe how nesprins are necessary in maintaining cellular architecture by acting as essential scaffolds and linkers at both the NE and other sub-cellular domains. More importantly, we speculate how nesprin mutations may disrupt tissue specific nesprin scaffolds and explain the tissue specific nature of many nesprin-associated diseases, including laminopathies. The eukaryotic cytoplasm contains three major composed of three α-helical bundles with a left- types of cytoskeletal filaments: Filamentous- handed twist, and its primary function is to actin (F-actin), microtubules (MTs) and provide docking sites for proteins and other intermediate filaments (IFs). These components higher order complexes (Refs 3, 4). Although are organised in a manner that provides the cell most SR proteins contain CHDs, some possess with an internal framework fundamental for motifs which can interact with other cytoskeletal many processes, such as controlling cellular components, allowing linkage of SR-associated shape, polarity, adhesion and migration, complexes to filamentous structures other than cytokinesis, inter- and intracellular F-actin. In addition, these motifs allow cross- Nesprins: from the nuclear envelope and beyond communication and trafficking of organelles, linking between different filaments and dynamic vesicles, proteins and RNA (Refs 1, 2).
    [Show full text]
  • Abnormal Nuclear Shape and Impaired Mechanotransduction in Emerin
    JCB: ARTICLE Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells Jan Lammerding,1 Janet Hsiao,2 P. Christian Schulze,1 Serguei Kozlov,3 Colin L. Stewart,3 and Richard T. Lee1 1Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA 02115 2HST Division, Massachusetts Institute of Technology, Cambridge, MA 02139 3Cancer and Developmental Biology Lab, National Cancer Institute, Frederick, MD 21702 mery-Dreifuss muscular dystrophy can be caused type cells in cellular strain experiments, and the integrity by mutations in the nuclear envelope proteins lamin of emerin-deficient nuclear envelopes appeared normal E A/C and emerin. We recently demonstrated that in a nuclear microinjection assay. Interestingly, expres- A-type lamin-deficient cells have impaired nuclear me- sion of mechanosensitive genes in response to mechani- chanics and altered mechanotransduction, suggesting cal strain was impaired in emerin-deficient cells, and two potential disease mechanisms (Lammerding, J., P.C. prolonged mechanical stimulation increased apoptosis in Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, emerin-deficient cells. Thus, emerin-deficient mouse em- C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113: bryo fibroblasts have apparently normal nuclear me- 370–378). Here, we examined the function of emerin on chanics but impaired expression of mechanosensitive nuclear mechanics and strain-induced signaling. Emerin- genes in response to strain, suggesting that emerin muta- deficient mouse embryo fibroblasts have
    [Show full text]
  • Produktinformation
    Produktinformation Diagnostik & molekulare Diagnostik Laborgeräte & Service Zellkultur & Verbrauchsmaterial Forschungsprodukte & Biochemikalien Weitere Information auf den folgenden Seiten! See the following pages for more information! Lieferung & Zahlungsart Lieferung: frei Haus Bestellung auf Rechnung SZABO-SCANDIC Lieferung: € 10,- HandelsgmbH & Co KG Erstbestellung Vorauskassa Quellenstraße 110, A-1100 Wien T. +43(0)1 489 3961-0 Zuschläge F. +43(0)1 489 3961-7 [email protected] • Mindermengenzuschlag www.szabo-scandic.com • Trockeneiszuschlag • Gefahrgutzuschlag linkedin.com/company/szaboscandic • Expressversand facebook.com/szaboscandic SYNE1 FISH Probe Catalog # : FA0178 規格 : [ 200 uL ] List All Specification Application Image Product Made to order FISH probes for identification of gene amplification using Fluorescent In Situ Hybridization (Cell) Description: Fluorescent In Situ Hybridization Technique. (Technology) Supplied DAPI Counterstain (1500 ng/mL ) 250 uL Product: Storage Store at 4°C in the dark. Instruction: Origin: Human Source: Genomic DNA Notice: We strongly recommend the customer to use FFPE FISH PreTreatment Kit 1 (Catalog #: KA2375 or KA2691) for the pretreatment of Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections. Regulation For research use only (RUO) Status: Applications Fluorescent In Situ Hybridization (Cell) Protocol Download Gene Information Entrez GeneID: 23345 Gene Name: SYNE1 Gene Alias: 8B,CPG2,DKFZp781J13156,FLJ30878,FLJ41140,KIAA0796,KIAA1262, KIAA1756,MYNE1,SCAR8 Gene spectrin repeat containing, nuclear envelope 1 Description: Omim ID: 608441, 610743 Gene Ontology: Hyperlink Gene Summary: This gene encodes a spectrin repeat containing protein expressed in skeletal and smooth muscle, and peripheral blood lymphocytes, that localizes to the nuclear membrane. Mutations in this gene have been associated with autosomal recessive spinocerebellar ataxia 8, also referred to as autosomal recessive cerebellar ataxia type 1 or recessive ataxia of Beauce.
    [Show full text]
  • Characterisation of Enaptin and Sun1, Two Novel Mammalian Nuclear Envelope Proteins
    Characterisation of Enaptin and Sun1, two novel mammalian nuclear envelope proteins INAUGURAL-DISSERTATION zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zü Köln vorgelegt von Padmakumar Velayuthan Chellammal aus Peruvilai, Indien Köln, 2004 Referees/Berichterstatter: Prof. Dr. Angelika A. Noegel Prof. Dr. Karin Schnetz Date of oral examination: 02.07.2004 Tag der mündlichen Prüfung The present research work was carried out under the supervision of Prof. Dr. Angelika A. Noegel, in the Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany. From August 2001 to July 2004. Diese Arbeit wurde von August 2001 bis Juli 2004 am Biochemischen Institut I der Medizinischen Fakultät der Universität zu Köln unter der Leitung von Prof. Dr. Angelika A. Noegel durchgeführt. First of all, I would like to express my heartiest gratitude to Prof.Dr.Noegel for giving me an opportunity to work in her group with Enaptin. Her positive attitude, constant encouragement and sustained interest in my project proved essential for my successful PhD work. I would like to thank Dr.Iakowos Karakesisoglou (Akis), our group leader for the excellent advice with my work, plentiful of encouragement and lots of motivation. He offered a hospitable environment in the lab with his friendly mannerisms and of course not to forget his sense of humour. I would also like to thank Dr.Elena Korenbaum with whom I started my PhD in the first year. It was a wonderful first year with her. It would be unfair if I don’t convey my thanks to Dr.Franciso Rivero, who helped me learn Confocal Microscopy and not the least, Spanish (all bad words).
    [Show full text]
  • Cloning and Characterization of Enaptin, a Novel Giant Actin-Binding Protein Connecting the Nucleus to the Actin Cytoskeleton
    Cloning and characterization of Enaptin, a novel giant actin-binding protein connecting the nucleus to the actin cytoskeleton INAUGURAL-DISSERTATION zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zü Köln vorgelegt von Sabu Abraham aus Thiruvaniyoor, Kerala, Indien Köln, 2004 Referees/Berichterstatter: Prof. Dr. Angelika A. Noegel Prof. Dr. Jens Brüning Date of oral examination: 08.07.2004 Tag der mündlichen Prüfung The present research work was carried out under the supervision of Prof. Dr. Angelika A. Noegel, in the Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany from August 2001 to July 2004. Diese Arbeit wurde von August 2001 bis Juli 2004 am Biochemischen Institut I der Medizinischen Fakultät der Universität zu Köln unter der Leitung von Prof. Dr. Angelika A. Noegel durchgeführt. To my beloved Pappa and Mummy Acknowledgements First of all, I would like to thank Prof. Dr. Angelika A Noegel for giving me an opportunity to work in her department and to do my PhD studies under her guidance. Her valuable guidance, creative suggestions, constructive criticism and constant encouragement during the course of work inspired and enabled me to complete this project. I would like to express here my deep sense of gratitude to her. I cannot find words to say thanks to our group leader Dr. Iakowos Karakesisoglou for his constant encouragement and discussions during the course of this study. His ‘story for a lesson’ approach always lifted my spirit up. It was wonderful to work with him. Thank you Akis. I would also like to thank Dr. Elena Korenbaum for guiding me in the first year of my study.
    [Show full text]
  • Snapshot: the Nuclear Envelope II Andrea Rothballer and Ulrike Kutay Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland
    SnapShot: The Nuclear Envelope II Andrea Rothballer and Ulrike Kutay Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland H. sapiens D. melanogaster C. elegans S. pombe S. cerevisiae Cytoplasmic filaments RanBP2 (Nup358) Nup358 CG11856 NPP-9 – – – Nup214 (CAN) DNup214 CG3820 NPP-14 Nup146 SPAC23D3.06c Nup159 Cytoplasmic ring and Nup88 Nup88 (Mbo) CG6819 – Nup82 SPBC13A2.02 Nup82 associated factors GLE1 GLE1 CG14749 – Gle1 SPBC31E1.05 Gle1 hCG1 (NUP2L1, NLP-1) tbd CG18789 – Amo1 SPBC15D4.10c Nup42 (Rip1) Nup98 Nup98 CG10198 Npp-10N Nup189N SPAC1486.05 Nup145N, Nup100, Nup116 Nup 98 complex RAE1 (GLE2) Rae1 CG9862 NPP-17 Rae1 SPBC16A3.05 Gle2 (Nup40) Nup160 Nup160 CG4738 NPP-6 Nup120 SPBC3B9.16c Nup120 Nup133 Nup133 CG6958 NPP-15 Nup132, Nup131 SPAC1805.04, Nup133 SPBP35G2.06c Nup107 Nup107 CG6743 NPP-5 Nup107 SPBC428.01c Nup84 Nup96 Nup96 CG10198 NPP-10C Nup189C SPAC1486.05 Nup145C Outer NPC scaffold Nup85 (PCNT1) Nup75 CG5733 NPP-2 Nup-85 SPBC17G9.04c Nup85 (Nup107-160 complex) Seh1 Nup44A CG8722 NPP-18 Seh1 SPAC15F9.02 Seh1 Sec13 Sec13 CG6773 Npp-20 Sec13 SPBC215.15 Sec13 Nup37 tbd CG11875 – tbd SPAC4F10.18 – Nup43 Nup43 CG7671 C09G9.2 – – – Centrin-21 tbd CG174931, CG318021 R08D7.51 Cdc311 SPCC1682.04 Cdc311 Nup205 tbd CG11943 NPP-3 Nup186 SPCC290.03c Nup192 Nup188 tbd CG8771 – Nup184 SPAP27G11.10c Nup188 Central NPC scaffold Nup155 Nup154 CG4579 NPP-8 tbd SPAC890.06 Nup170, Nup157 (Nup53-93 complex) Nup93 tbd CG7262 NPP-13 Nup97, Npp106 SPCC1620.11, Nic96 SPCC1739.14 Nup53(Nup35, MP44) tbd CG6540 NPP-19 Nup40 SPAC19E9.01c
    [Show full text]
  • N Karry: the KASH Domain Family of Cargo-Specific Cytoskeletal Adaptor Proteins Daniel A
    Review articles KASH ‘n Karry: the KASH domain family of cargo-specific cytoskeletal adaptor proteins Daniel A. Starr1 and Janice A. Fischer2* Summary membrane interacts with the cytoskeleton to position nuclei A diverse family of proteins has been discovered with a within the cell. As the nucleus is probably the easiest cargo to small C-terminal KASH domain in common. KASH domain proteins are localized uniquely to the outer visualize and follow in real time, nuclear positioning makes an nuclear envelope, enabling their cytoplasmic extensions excellent model to study cargo adaptors. Also, nuclear to tether the nucleus to actin filaments or microtubules. migration through the cytoplasm and nuclear anchorage KASH domains are targeted to the outer nuclear envelope within a polarized cell are essential processes for the develop- by SUN domains of inner nuclear envelope proteins. ment of nearly all eukaryotes (for review see Refs. 1–3). Our Several KASH protein genes were discovered as mutant story begins with a group of Drosophila and C. elegans alleles in model organisms with defects in developmen- tally regulated nuclear positioning. Recently, KASH-less mutants with mispositioned nuclei (Fig. 1). As described below, isoforms have been found that connect the cytoskeleton molecular characterization of these mutants has identified a to organelles other than the nucleus. A widened view of class of outer nuclear membrane components called KASH these proteins is now emerging, where KASH proteins proteins. Recent evidence has shown that certain isoforms of and their KASH-less counterparts are cargo-specific adaptors that not only link organelles to the cytoskeleton these proteins adapt cargos other than the nucleus.
    [Show full text]
  • Recovery of Stem Cell Proliferation by Low Intensity Vibration Under Simulated Microgravity Requires LINC Complex
    www.nature.com/npjmgrav ARTICLE OPEN Recovery of stem cell proliferation by low intensity vibration under simulated microgravity requires LINC complex H. Touchstone1,R.Bryd1, S. Loisate1, M. Thompson1, S. Kim2, K. Puranam2, A. N. Senthilnathan2,X.Pu3, R. Beard 3, J. Rubin2, J. Alwood 4, J. T. Oxford3 and G. Uzer1 Mesenchymal stem cells (MSC) rely on their ability to integrate physical and spatial signals at load bearing sites to replace and renew musculoskeletal tissues. Designed to mimic unloading experienced during spaceflight, preclinical unloading and simulated microgravity models show that alteration of gravitational loading limits proliferative activity of stem cells. Emerging evidence indicates that this loss of proliferation may be linked to loss of cellular cytoskeleton and contractility. Low intensity vibration (LIV) is an exercise mimetic that promotes proliferation and differentiation of MSCs by enhancing cell structure. Here, we asked whether application of LIV could restore the reduced proliferative capacity seen in MSCs that are subjected to simulated microgravity. We found that simulated microgravity (sMG) decreased cell proliferation and simultaneously compromised cell structure. These changes included increased nuclear height, disorganized apical F-actin structure, reduced expression, and protein levels of nuclear lamina elements LaminA/C LaminB1 as well as linker of nucleoskeleton and cytoskeleton (LINC) complex elements Sun-2 and Nesprin-2. Application of LIV restored cell proliferation and nuclear proteins LaminA/C and Sun-2. An intact LINC function was required for LIV effect; disabling LINC functionality via co-depletion of Sun-1, and Sun-2 prevented rescue of cell proliferation by LIV. Our findings show that sMG alters nuclear structure and leads to decreased cell proliferation, but does not diminish LINC complex mediated mechanosensitivity, suggesting LIV as a potential candidate to combat sMG-induced proliferation loss.
    [Show full text]
  • Product Data Sheet Purified Anti-Nesprin-1
    Version: 2 Revision Date: 2015-08-20 Product Data Sheet Purified anti-Nesprin-1 Catalog # / Size: 902701 / 100 µl Previously: Covance Catalog# PRB-439P Clone: Poly19027 Isotype: Rabbit Polyclonal Immunogen: This antibody was raised against amino acids 1-428 of recombinantly expressed rat nesprin-1-alpha, and thus recognizes all nesprin-1 forms, alpha and beta Reactivity: Rat, Mouse, Human, Primate, Dog Preparation: The antibody was purified by affinity chromatography. Formulation: Phosphate-buffered solution. Concentration: 1 mg/ml Storage: The antibody solution should be stored undiluted between 2°C and 8°C. Please note the storage condition for this antibody has been changed from -20°C to between 2°C and 8°C. You can also check your vial or your CoA to find the most accurate storage condition for this antibody. Applications: Staining of Nesprin-1 (clone Poly19027) on formalin fixed paraffin Applications: IHC, WB, IF, IP embedded human skeletal muscle tissue at 1/500. Recommended Usage: Each lot of this antibody is quality control tested by immunohistochemical staining. The optimal working dilution should be determined for each specific assay condition. • WB: 1:2,000* • IF: 1:500 • IP: 1:500 • IHC: 1:500-1:1,000 Tissue Sections: Formalin-fixed, paraffin-embedded tissues & frozen sections Pretreatment: For optimal staining, the sections should be pretreated with an antigen unmasking solution such as citrate buffer retrieval solution (Cat. No. 928501, 928601). Incubation: 24 hours at 2-8°C Application Notes: This antibody is effective in immunoblotting (WB), immunocytochemistry (ICC), immunoprecipitation (IP) and immunohistochemistry (IHC). *Predicted MW = 120 kD Positive Control: human skeletal muscle Application References: 1.
    [Show full text]
  • High-Throughput Detection, Annotation, and Interpretation Of
    High-throughput detection, annotation, and interpretation of genomic variants using next-generation sequencing data by Dewey J. Kim A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. Baltimore, Maryland Oct, 2015 © Dewey J. Kim 2015 All rights reserved Abstract Recent advances in sequencing technology have made it feasible to use next- generation sequencing (NGS) to characterize the genomic landscape of an indi- vidual. Unlike microarrays, which are usually only capable of detecting variants that have been previously discovered in a population, NGS is capable of discern- ing both common and rare de novo variants. Sequencing studies that involve the analysis of rare variants in human disease typically follow three steps: variant calling, where variants in NGS data are iden- tified, variant annotation, where biologically relevant features are attached to each variant, and variant interpretation, where statistical and machine learning meth- ods are used to prioritize putative functional variants. In this thesis, I attempt to apply and improve these methods in the context of cancer and schizophrenia. Recent matched tumor/normal whole exome sequencing studies, coupled with current variant calling tools, have generated large sets of high-confidence genomic variants. A significant proportion of these variants are missense variants of un- known impact. To increase the speed and efficiency of annotating these variants, ii ABSTRACT I helped in the creation of a database of 86 precomputed disease-relevant fea- tures for all possible missense variants in the human exome. This tool allows for near-instantaneous annotation of any variant dataset.
    [Show full text]
  • Human Nesprin-1 (SYNE1) ELISA Kit
    Product Datasheet Human Nesprin-1 (SYNE1) ELISA Kit Catalog No: #EK6735 Package Size: #EK6735-1 48T #EK6735-2 96T Orders: [email protected] Support: [email protected] Description Product Name Human Nesprin-1 (SYNE1) ELISA Kit Brief Description ELISA Kit Applications ELISA Species Reactivity Human (Homo sapiens) Other Names RP1-130E4.2; 8B; C6orf98; CPG2; DKFZp781J13156; EDMD4; FLJ30878; FLJ41140; KIAA0796; KIAA1262; KIAA1756; MYNE1; SCAR8; dJ45H2.2; OTTHUMP00000017438|enaptin|myocyte nuclear envelope protein 1|nesprin Accession No. Q8NF91 Storage The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37C for 4 and 7 days, and compare O.D.values of the kit kept at 37C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37C can be considered as 6 months at 2 - 8C, which means 7 days at 37C equaling 12 months at 2 - 8C). Application Details Detect Range:0.312-20 ng/mL Sensitivity: 0.105 ng/mL Sample Type:Serum, Plasma, Other biological fluids Sample Volume: 1-200 µL Assay Time:1-4.5h Detection wavelength:450 nm Product Description Detection Method:SandwichTest principle:This assay employs a two-site sandwich ELISA to quantitate SYNE1 in samples. An antibody specific for SYNE1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anySYNE1 present is bound by the immobilized antibody.
    [Show full text]