DOCSLIB.ORG

Telomeric Nucleic Acids: C-Strand Structure and a Telomerase RNA Mutant Shawn Cameron Ahmed Iowa State University

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Telomeric Nucleic Acids: C-Strand Structure and a Telomerase RNA Mutant Shawn Cameron Ahmed Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1994 Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant Shawn Cameron Ahmed Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biophysics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ahmed, Shawn Cameron, "Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant " (1994). Retrospective Theses and Dissertations. 10529. https://lib.dr.iastate.edu/rtd/10529 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly firom the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from ai^ of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproductioiL In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9518349 Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant Ahmed, Shawn Cameron, Ph.D. Iowa State University, 1994 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant by Shawn Cameron Ahmed A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Zoology and Genetics Interdepartmental Major: Molecular, Cellular and Developmental Biology Approved: Signature was redacted for privacy. In Charge of Mai.or Work Signature was redacted for privacy. For the Infta'departmental Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1994 ii Dedicated to my parents, l\/lohammed and Shirley iii TABLE OF CONTENTS ABSTRACT iv INTRODUCTION 1 CHAPTER 1: FORMATION OF NOVEL HAIRPIN STRUCTURES BY TELOMERIC C-STRAND OLIGONUCLEOTIDES 8 CHAPTER 2: HUMAN TELOMERIC C-STRAND TETRAPLEXES 18 CHAPTER 3: tgifl, A TELOMERASE RNA MUTANT WITH SHORT TELOMERES 30 GENERAL CONCLUSIONS 48 ACKNOWLEDGEMENTS 54 iv ABSTRACT Telomeres, the ends of linear chromosomes, are composed of simple tandem repeats which are usually G-C rich. Telomeres are essential for chromosome stability, organizing the nuclear architecture and ensuring complete replication of the chromosomal terminus. To understand how telomeres carry out these fundamental cellular roles, one must understand the structural and dynamic properties of telomeric repeat sequences. Structural and genetic approaches were taken to learn more about telomeric nucleic acids. The structural portion of my research concerned an unusual DNA structure formed by the C- rich strand of telomeric DNA. Telomeric C-strand sequences form non-Watson-Crick structures in supercoiled plasmids at low pH. Absorbance thermal denaturation, chemical modification and non- denaturing gel electrophoresis showed that telomeric C-strand oligonucleotides form stable structures at low pH. nuclear Overhauser effects indicated that these structures were four-stranded. In addition, these four-stranded C-structures were shown to mediate recognition and binding of identical nucleic acid sequences. Thus, a novel nucleic acid dimerization motif was discovered. The genetic portion of my research concerned analysis of a Tetrahymena mutant with short telomeres. This mutant was heterozygous for a telomerase RNA mutation. Telomerase is a ribonucleoprotein that uses its RNA component as a template for addition of telomeric repeats to chromosome termini. Therefore, telomerase is involved in telomere length regulation, a process that has been implicated in both aging and cancer. The mutant telomerase RNA gene caused telomere shortening when introduced into wildtype cells, and thus identifies a functionally important domain of the telomerase RNA. Although mutant telomerase activity was indistinguishable from wildtype activity in vitro, cells expressing high levels of the mutant telomerase RNA exhibited lethal phenotypes that were due to the presence of very short telomeres. 1 INTRODUCTION Thesis Organization. This introduction is meant to provide a broad general perspective of current l<nowledge in the field of telomeres as it relates to my research. The main body of this thesis is composed of three papers, and a conclusion at the end of this thesis touches on the fruits of my efforts and discusses research topics that may be worthy of further investigation. References for the introduction and conclusion are cited after the conclusion. The first two papers have been published in peer-reviewed journals. These two papers are the result of my investigations into telomeric C-strand structure. The third paper is a 'manuscript in preparation' and discusses my efforts to understand a telomerase RNA mutation that I discovered. All three papers have different formats because they either have been or will be published in different journals. References are given at the end of each paper. 1 am the primary author on each paper and personally completed most or all of the research described. My research was conducted with the guidance of Eric Henderson, the principal investigator. Agustin Kintanar provided invaluable assistance with NMR spectroscopy in paper 2. Mohammed Al-Anazi and Hong Sheng produced several of the mutants described in paper 3. Telomeres. Telomeres, chromatin domains at the ends of eukaryotic chromosomes, protect chromosomes from degradation and end-to-end fusion, presumably by burying the 'end' in a specialized nucleoprotein complex (Blackburn, 1991; Greider, 1990). Telomeres also help organize a chromosome's architecture within the nucleus and often cluster together at the nuclear periphery (Blackburn and Szostack, 1984). Chromosome ends without telomeres are unstable, and telomere loss often results in chromosome loss (Sandell and Zakian, 1993). Thus, telomeres are essential for genome viability and integrity. Conserved molecular mechanisms by which telomeres accomplish this task probably developed very early in eukaryotic evolution. Telomeric DNA. In most eukaryotes, telomeric DNA is composed of simple tandem repeats, though the sequence of these repeats can vary from organism to organism (Zakian, 1989). Most telomeric repeats have an asymmetric distribution of guanine and cytosine resulting in one strand being G-rich and the other C-rich. The G-strand always run 5' to 3' towards the end of the chromosome and extends as a single strand by about two repeats (Figure 1) (Henderson and Blackburn, 1989; Wellinger, et al., 1993). Evolutionary conservation of this strand composition bias suggests that it is important to telomere structure and function. Curiously, both strands of telomeric DNA can form their own unusual structures. •••^•AACCCC^ •••TTGGGGTTGGGGTTGGGG I simple, repetitive G-C rich sequences Figure 1. The Tetrahymena telomeric terminus has a 2 repeat 3' overhang. 2 Unusual DNA structures. DNA has the ability to adopt a variety of structures. Most DNA in a cell is double-stranded B-form DNA. However, double-stranded DNA can adopt alternative conformations such as A- and Z-DNA. In addition, three- and four-stranded DNA structures have been shown to exist. These less abundant DNA structures probably have specific biological functions (Rich, 1993). Methods for determining the biological function of an unusual DNA structure vary, but, in general, studies investigate sequences which form unusual structures and proteins that interact with these structures. Ultimately, mutational analysis of sequences which form such structures and of proteins that interact with them will enable us to understand their biological role. Telomeric DNA has been a useful 'model system' for studying unusual DNA structures. G-strand structures. In the presence of monovalent cations, the G-strand of telomeric DNA was shown to form unusual DNA structures (Henderson, et al., 1987) that were four- stranded (Williamson, et al., 1989; Sen and Gilbert, 1990). Since then, several groups have clearly demonstrated that the ends
Load more

Recommended publications
  • Specific Loop Modifications of the Thrombin Binding Aptamer Trigger the Formation of Parallel Structures
    “Specific loop modifications of the thrombin binding aptamer trigger the formation of parallel structures” Aviñó, A., Portella, G., Ferreira, R., Gargallo, R., Mazzini, S., Gabélica, V., Orozco, M., Eritja, R. The FEBS J., 281(4), 1085-1099 (2014). PMID: 24304855, doi: 10.1111/febs.12670 SPECIFIC LOOP MODIFICATIONS OF THE THROMBIN BINDING APTAMER TRIGGER THE FORMATION OF PARALLEL STRUCTURES Anna Aviñó1,#,Guillem Portella2,3,#, Ruben Ferreira1, Raimundo Gargallo5, Stefania Mazzini6, Valerie Gabélica7, Modesto Orozco,2,3,4*and Ramon Eritja1* 1Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Jordi Girona 18-26 08034 Barcelona, Spain. 2Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona, Spain 3Joint IRB-BSC Program in Computational Biology, Barcelona, Spain 4Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, University of Barcelona, 08028 Barcelona, Spain 5Department of Analytical Chemistry, University of Barcelona, 08028 Barcelona, Spain 6Department of Food, Environmental and Nutritional Sciences (DEFENS), Section of Chemical and Biomolecular Sciences, University of Milan, Via Celoria 2, 20133 Milan, Italy 7Physical Chemistry and Mass Spectrometry Laboratory, Department of Chemistry, University of Liège, Belgium. (Present addresses: (1) Univ. Bordeaux, IECB - ARNA laboratory, F-33600 Pessac, France, (2) INSERM, U869 - ARNA laboratory, F-33000 Bordeaux, France. * To whom correspondence should
    [Show full text]
  • Identification of Foxr2 As an Oncogene in Medulloblastoma
    Published OnlineFirst March 5, 2014; DOI: 10.1158/0008-5472.CAN-13-1523 Cancer Tumor and Stem Cell Biology Research Identification of FoxR2 as an Oncogene in Medulloblastoma Hideto Koso1,2, Asano Tsuhako1, Eli Lyons1, Jerrold M. Ward2, Alistair G. Rust3, David J. Adams3, Nancy A. Jenkins2,4, Neal G. Copeland2,4, and Sumiko Watanabe1 Abstract Medulloblastoma is the most common pediatric brain tumor, and in 25% of cases, it is driven by aberrant activation of the Sonic Hedgehog (SHH) pathway in granule neuron precursor (GNP) cells. In this study, we identified novel medulloblastoma driver genes through a transposon mutagenesis screen in the developing brain of wild-type and Trp53 mutant mice. Twenty-six candidates were identified along with established driver genes such as Gli1 and Crebbp. The transcription factor FoxR2, the most frequent gene identified in the screen, is overexpressed in a small subset of human medulloblastoma of the SHH subtype. Tgif2 and Alx4, 2 new putative oncogenes identified in the screen, are strongly expressed in the SHH subtype of human medulloblas- toma. Mutations in these two genes were mutually exclusive with mutations in Gli1 and tended to cooccur, consistent with involvement in the SHH pathway. Notably, Foxr2, Tgif2, and Alx4 activated Gli-binding sites in cooperation with Gli1, strengthening evidence that they function in SHH signaling. In support of an oncogenic function, Foxr2 overexpression transformed NIH3T3 cells and promoted proliferation of GNPs, the latter of which was also observed for Tgif2 and Alx4. These findings offer forward genetic and functional evidence associating Foxr2, Tgif2, and Alx4 with SHH subtype medulloblastoma.
    [Show full text]
  • TGIF2 Monoclonal Antibody (M06), Clone 6A8
    TGIF2 monoclonal antibody (M06), clone 6A8 Catalog # : H00060436-M06 規格 : [ 100 ug ] List All Specification Application Image Product Mouse monoclonal antibody raised against a partial recombinant Western Blot (Cell lysate) Description: TGIF2. Immunogen: TGIF2 (NP_068581, 131 a.a. ~ 236 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa. Sequence: SMPLHSGQGEKPAAPFPRGELESPKPLVTPGSTLTLLTRAEAGSPTGG LFNTPPPTPPEQDKEDFSSFQLLVEVALQRAAEMELQKQQDPSLPLLHT enlarge PIPLVSENP Western Blot (Recombinant protein) Host: Mouse Immunofluorescence Reactivity: Human Isotype: IgG2a Kappa Quality Control Antibody Reactive Against Recombinant Protein. Testing: enlarge Sandwich ELISA (Recombinant protein) enlarge ELISA Western Blot detection against Immunogen (37.4 KDa) . Storage Buffer: In 1x PBS, pH 7.4 Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: MSDS: Download Datasheet: Download Applications Western Blot (Cell lysate) Page 1 of 3 2020/3/20 TGIF2 monoclonal antibody (M06), clone 6A8 Western Blot analysis of TGIF2 expression in IMR-32 ( Cat # L008V1 ). Protocol Download Western Blot (Recombinant protein) Protocol Download Immunofluorescence enlarge this image Immunofluorescence of monoclonal antibody to TGIF2 on HeLa cell. [antibody concentration 10 ug/ml] Sandwich ELISA (Recombinant protein) Detection limit for recombinant GST tagged TGIF2 is approximately 0.3ng/ml as a capture antibody. Protocol Download ELISA Gene Information Entrez GeneID: 60436 GeneBank NM_021809 Accession#: Protein NP_068581 Accession#: Gene Name: TGIF2 Gene Alias: - Page 2 of 3 2020/3/20 Gene TGFB-induced factor homeobox 2 Description: Omim ID: 607294 Gene Ontology: Hyperlink Gene Summary: The protein encoded by this gene is a DNA-binding homeobox protein and a transcriptional repressor. The encoded protein appears to repress transcription by recruiting histone deacetylases to TGF beta- responsive genes.
    [Show full text]
  • Extensive Evaluation of Force Fields for G-Quadruplexes
    Extensive Evaluation of Force Fields for G-Quadruplexes Na Li1, Tong Zhu1,2* 1Shanghai Engineering Research Center of Molecular Therapeutics & New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, People’s Republic of China. 2NYU-ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai, People’s Republic of China ABSTRACT G-Quadruplexes (GQs), folded by guanine-rich nucleic acid sequences, involve in gene expression processes and closely associated with the formation of tumors. So far, GQ has drawn widespread attention for its notable application of serving as potential anti-cancer target. Recently, theoretical studies for GQs have achieved significant progress, most of which are inseparable from molecular dynamics (MD) simulation. As a necessary tool to explore dynamics behavior of molecules, MD simulations strictly depend on force field parameters, which is a sticking point to obtain accurate results. Currently, many force fields for nucleic acids have been developed, but none of them have been accepted widely for GQs. In this paper, we selected five popular force fields, which are parmbsc0, parmbsc1, OL15, Drude2017 and AMOEBANUC17, and conducted explicit-solvent MD simulations on two DNA GQs respectively. We evaluated these force fields from many aspects in detail. Meanwhile, we compared conformational energy using quantum chemistry calculations. With the comprehensive evaluation, Drude2017 achieved better description for GQs, which we suggest that using Drude2017 force field should be taken into account first when investigating GQs by MD simulation. * To whom correspondence should be addressed. Email: [email protected]; [email protected] INTRODUCTION G-Quadruplexes (GQs)1 are folded by guanine-rich nucleic acid sequences.
    [Show full text]
  • Structure and Function of Multimeric G-Quadruplexes
    molecules Review Structure and Function of Multimeric G-Quadruplexes Sofia Kolesnikova 1,2 and Edward A. Curtis 1,* 1 The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 166 10 Prague, Czech Republic 2 Department of Biochemistry and Microbiology, University of Chemistry and Technology, 166 28 Prague, Czech Republic * Correspondence: [email protected] Received: 13 August 2019; Accepted: 22 August 2019; Published: 24 August 2019 Abstract: G-quadruplexes are noncanonical nucleic acid structures formed from stacked guanine tetrads. They are frequently used as building blocks and functional elements in fields such as synthetic biology and also thought to play widespread biological roles. G-quadruplexes are often studied as monomers, but can also form a variety of higher-order structures. This increases the structural and functional diversity of G-quadruplexes, and recent evidence suggests that it could also be biologically important. In this review, we describe the types of multimeric topologies adopted by G-quadruplexes and highlight what is known about their sequence requirements. We also summarize the limited information available about potential biological roles of multimeric G-quadruplexes and suggest new approaches that could facilitate future studies of these structures. Keywords: G-quadruplex; dimer; tetramer; multimer; oligomer; telomere; promoter; R-loop; DNA:RNA hybrid 1. Introduction The B-form double helix is the most well known nucleic acid structure, but it is not the only one. Other examples include double helices with geometries that differ from that of classical B-form DNA, such as Z-DNA [1], triple helices [2], and even four-stranded structures in which canonical A-T and C-G base pairs are absent [3,4].
    [Show full text]
  • Rapid Evolution of Mammalian X-Linked Testis-Expressed Homeobox Genes
    Copyright 2004 by the Genetics Society of America DOI: 10.1534/genetics.103.025072 Rapid Evolution of Mammalian X-Linked Testis-Expressed Homeobox Genes Xiaoxia Wang and Jianzhi Zhang1 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109 Manuscript received November 26, 2003 Accepted for publication February 11, 2004 ABSTRACT Homeobox genes encode transcription factors that function in various developmental processes and are usually evolutionarily conserved in their sequences. However, two X-chromosome-linked testis-expressed homeobox genes, one from rodents and the other from fruit flies, are known to evolve rapidly under positive Darwinian selection. Here we report yet another case, from primates. TGIFLX is an X-linked homeobox gene that originated by retroposition of the autosomal gene TGIF2, most likely in a common ancestor of rodents and primates. While TGIF2 is ubiquitously expressed, TGIFLX is exclusively expressed in adult testis. A comparison of the TGIFLX sequences among 16 anthropoid primates revealed a signifi- cantly higher rate of nonsynonymous nucleotide substitution (dN) than synonymous substitution (dS), strongly suggesting the action of positive selection. Although the high dN/dS ratio is most evident outside ف the homeobox, the homeobox has a dN/dS of 0.89 and includes two codons that are likely under selection. Furthermore, the rate of radical amino acid substitutions that alter amino acid charge is significantly greater than that of conservative substitutions, suggesting that the selection promotes diversity of the protein charge profile. More interestingly, an analysis of 64 orthologous homeobox genes from humans and mice shows substantially higher rates of amino acid substitution in X-linked testis-expressed genes than in other genes.
    [Show full text]
  • 6 Signaling and BMP Antagonist Noggin in Prostate Cancer
    [CANCER RESEARCH 64, 8276–8284, November 15, 2004] Bone Morphogenetic Protein (BMP)-6 Signaling and BMP Antagonist Noggin in Prostate Cancer Dominik R. Haudenschild, Sabrina M. Palmer, Timothy A. Moseley, Zongbing You, and A. Hari Reddi Center for Tissue Regeneration and Repair, Department of Orthopedic Surgery, School of Medicine, University of California, Davis, Sacramento, California ABSTRACT antagonists has recently been discovered. These are secreted proteins that bind to BMPs and reduce their bioavailability for interactions It has been proposed that the osteoblastic nature of prostate cancer with the BMP receptors. Extracellular BMP antagonists include nog- skeletal metastases is due in part to elevated activity of bone morphoge- gin, follistatin, sclerostatin, chordin, DCR, BMPMER, cerberus, netic proteins (BMPs). BMPs are osteoinductive morphogens, and ele- vated expression of BMP-6 correlates with skeletal metastases of prostate gremlin, DAN, and others (refs. 11–16; reviewed in ref. 17). There are cancer. In this study, we investigated the expression levels of BMPs and several type I and type II receptors that bind to BMPs with different their modulators in prostate, using microarray analysis of cell cultures affinities. BMP activity is also regulated at the cell membrane level by and gene expression. Addition of exogenous BMP-6 to DU-145 prostate receptor antagonists such as BAMBI (18), which acts as a kinase- cancer cell cultures inhibited their growth by up-regulation of several deficient receptor. Intracellularly, the regulation of BMP activity at cyclin-dependent kinase inhibitors such as p21/CIP, p18, and p19. Expres- the signal transduction level is even more complex. There are inhib- sion of noggin, a BMP antagonist, was significantly up-regulated by itory Smads (Smad-6 and Smad-7), as well as inhibitors of inhibitory BMP-6 by microarray analysis and was confirmed by quantitative reverse Smads (AMSH and Arkadia).
    [Show full text]
  • The DNA Sequence and Comparative Analysis of Human Chromosome 20
    articles The DNA sequence and comparative analysis of human chromosome 20 P. Deloukas, L. H. Matthews, J. Ashurst, J. Burton, J. G. R. Gilbert, M. Jones, G. Stavrides, J. P. Almeida, A. K. Babbage, C. L. Bagguley, J. Bailey, K. F. Barlow, K. N. Bates, L. M. Beard, D. M. Beare, O. P. Beasley, C. P. Bird, S. E. Blakey, A. M. Bridgeman, A. J. Brown, D. Buck, W. Burrill, A. P. Butler, C. Carder, N. P. Carter, J. C. Chapman, M. Clamp, G. Clark, L. N. Clark, S. Y. Clark, C. M. Clee, S. Clegg, V. E. Cobley, R. E. Collier, R. Connor, N. R. Corby, A. Coulson, G. J. Coville, R. Deadman, P. Dhami, M. Dunn, A. G. Ellington, J. A. Frankland, A. Fraser, L. French, P. Garner, D. V. Grafham, C. Grif®ths, M. N. D. Grif®ths, R. Gwilliam, R. E. Hall, S. Hammond, J. L. Harley, P. D. Heath, S. Ho, J. L. Holden, P. J. Howden, E. Huckle, A. R. Hunt, S. E. Hunt, K. Jekosch, C. M. Johnson, D. Johnson, M. P. Kay, A. M. Kimberley, A. King, A. Knights, G. K. Laird, S. Lawlor, M. H. Lehvaslaiho, M. Leversha, C. Lloyd, D. M. Lloyd, J. D. Lovell, V. L. Marsh, S. L. Martin, L. J. McConnachie, K. McLay, A. A. McMurray, S. Milne, D. Mistry, M. J. F. Moore, J. C. Mullikin, T. Nickerson, K. Oliver, A. Parker, R. Patel, T. A. V. Pearce, A. I. Peck, B. J. C. T. Phillimore, S. R. Prathalingam, R. W. Plumb, H. Ramsay, C. M.
    [Show full text]
  • Link Between the Causative Genes of Holoprosencephaly: Zic2 Directly
    www.nature.com/scientificreports OPEN Link between the causative genes of holoprosencephaly: Zic2 directly regulates Tgif1 expression Received: 20 October 2017 Akira Ishiguro1,2, Minoru Hatayama1,3, Maky I. Otsuka1 & Jun Aruga1,3 Accepted: 15 January 2018 One of the causal genes for holoprosencephaly (HPE) is ZIC2 (HPE5). It belongs to the zinc fnger Published: xx xx xxxx protein of the cerebellum (Zic) family of genes that share a C2H2-type zinc fnger domain, similar to the GLI family of genes. In order to clarify the role of Zic2 in gene regulation, we searched for its direct target genes using chromatin immunoprecipitation (ChIP). We identifed TGIF1 (HPE4), another holoprosencephaly-causative gene in humans. We identifed Zic2-binding sites (ZBS) on the 5′ fanking region of Tgif1 by in vitro DNA binding assays. ZBS were essential for Zic2-dependent transcriptional activation in reporter gene assays. Zic2 showed a higher afnity to ZBS than GLI-binding sequences. Zic2-binding to the cis-regulatory element near the Tgif1 promoter may be involved in the mechanism underlying forebrain development and incidences of HPE. Holoprosencephaly (HPE) is known as a common forebrain defect in human development1,2. At least 13 chro- mosomal loci are associated with nonsyndromic HPE2,3. SHH, ZIC2, SIX3, and TGIF1 have been identifed to be four causative genes in the HPE loci (HPE3, HPE5, HPE2, and HPE4, respectively) and have been investigated with respect to clinical spectrum4 and genetic interactions5. Among the major HPE-associated genes, the roles of TGIF1 and ZIC2 in forebrain development have been elusive until recently1. However, recent studies have revealed clues as to its roles in forebrain development.
    [Show full text]
  • TGIF2 Human Sirna Oligo Duplex (Locus ID 60436
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for SR311824 TGF beta induced factor 2 (TGIF2) Human siRNA Oligo Duplex (Locus ID 60436) Product data: Product Type: siRNA Oligo Duplexes Purity: HPLC purified Quality Control: Tested by ESI-MS Sequences: Available with shipment Stability: One year from date of shipment when stored at -20°C. # of transfections: Approximately 330 transfections/2nmol in 24-well plate under optimized conditions (final conc. 10 nM). Note: Single siRNA duplex (10nmol) can be ordered. RefSeq: NM_001199513, NM_001199514, NM_001199515, NM_021809 Components: TGIF2 (Human) - 3 unique 27mer siRNA duplexes - 2 nmol each (Locus ID 60436) Included - SR30004, Trilencer-27 Universal Scrambled Negative Control siRNA Duplex - 2 nmol Included - SR30005, RNAse free siRNA Duplex Resuspension Buffer - 2 ml Summary: The protein encoded by this gene is a DNA-binding homeobox protein and a transcriptional repressor, which appears to repress transcription by recruiting histone deacetylases to TGF beta-responsive genes. This gene is amplified and over-expressed in some ovarian cancers. Alternative splicing results in multiple transcript variants. A related pseudogene has been identified on chromosome 1. Read-through transcription also exists between this gene and the neighboring downstream C20orf24 (chromosome 20 open reading frame 24) gene. [provided by RefSeq, Dec 2010] This product
    [Show full text]
  • Thermodynamic Characterization of Tetraplex Dna Structures
    THERMODYNAMIC CHARACTERIZATION OF TETRAPLEX DNA STRUCTURES by BYUL KIM A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University of Toronto © Copyright by Byul Kim 2018 ABSTRACT Thermodynamic Characterization of Tetraplex DNA Structures Byul Kim Doctor of Philosophy Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University of Toronto 2018 The role of counterion condensation as a dominant force governing the stability of DNA duplexes and triplexes is well established. In contrast, the effect of counterion condensation on the stability of G-quadrupex conformations is poorly understood. Unlike other ordered nucleic acid structures, G-quadruplexes exhibit a specific binding of counterions (typically, Na+ or K+) which are buried inside the central cavity and coordinated to the O6 carbonyls of the guanines forming the G-quartets. While it has been known that the G-quadruplex-to-coil transition temperature, TM, increases with an increase in the concentration of the stabilizing ion, the contributions of the specific (coordination in the central cavity) and nonspecific (condensation) ion binding have not been resolved. In the first part of the work performed in this dissertation, we used G-quadruplexes formed by four different sequences derived from the human telomeric region, c-MYC, and VEGF genes to separate the two ionic contributions. We studied the change + + in TM of preformed G-quadruplexes following the addition of nonstabilizing ions Li , Cs , and TMA+ (tetramethylammonium). Our data suggest that the stabilizing action of cations on the G- quadruplex conformation is, primarily, due to the central ions which act as specifically bound II ligands.
    [Show full text]
  • DNA Sequences That Interfere with Transcription: Implications for Genome Function and Stability † ‡ † Boris P
    Review pubs.acs.org/CR DNA Sequences That Interfere with Transcription: Implications for Genome Function and Stability † ‡ † Boris P. Belotserkovskii, Sergei M. Mirkin, and Philip C. Hanawalt*, † Department of Biology, Stanford University, Stanford, California 94305, United States ‡ Department of Biology, Tufts University, Medford, Massachusetts 02155, United States 1. INTRODUCTION The primary role of DNA-dependent RNA synthesis, or transcription, is to create components for the cellular machinery. The nascent RNA product of transcription is released from the DNA template and either serves as an intermediate message for protein synthesis or is used directly, as in the case of rRNA, tRNA, and various types of regulatory RNAs. Recently, a growing number of examples suggest that transcription per se, rather than its released product, could play a regulatory role in gene function or as a trigger for genomic modifications. The latter scenario is commonly attributed to anomalous progression of the RNA polymerase (RNAP), such as pausing CONTENTS or termination and/or retaining rather than releasing the nascent transcript. This has been implicated in class-switch 1. Introduction 8620 recombination and somatic hypermutation (reviewed in refs 1, − 2. Transcription Blockage Mechanisms 8621 2), telomere maintenance,3 and replication initiation.4 9 3. Effects of Unusual DNA Structures and DNA/RNA Anomalous transcription elongation has also been linked to Complexes on Transcription 8622 various deleterious phenomena, such as genomic instabilities, 3.1. General Introduction to DNA Structure and transcription−replication collisions, and transcription defi- DNA Supercoiling 8622 ciency in some hereditary human disorders (reviewed in refs 3.2. Overview of Unusual Structures and Their 10−14).
    [Show full text]