DOCSLIB.ORG

(12) United States Patent (10) Patent No.: US 8,377,657 B1 Shuber (45) Date of Patent: Feb

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(12) United States Patent (10) Patent No.: US 8,377,657 B1 Shuber (45) Date of Patent: Feb US008377657B1 (12) United States Patent (10) Patent No.: US 8,377,657 B1 Shuber (45) Date of Patent: Feb. 19, 2013 (54) PRMERS FOR ANALYZING METHYLATED (56) References Cited SEQUENCES AND METHODS OF USE THEREOF U.S. PATENT DOCUMENTS 6,200,756 B1 3/2001 Herman et al. 7,790,385 B2 9/2010 Kutyavin et al. (75) Inventor: Anthony P. Shuber, Mendon, MA (US) 2005.0009047 A1 1/2005 Erlander et al. .................. 435/6 2011, 0008.845 A1 1/2011 Kim et al. OTHER PUBLICATIONS (73) Assignee: Predictive Biosciences Corporation, Lexington, MA (US) Herman et al., “Methylation-speciifc PCR: A novel PCR assay for methylation status of CpG islands.” PNAS, 93:9821-9826 (1996). Anglim et al., “DNA methylation-based biomarkers for early detec (*) Notice: Subject to any disclaimer, the term of this tion of non-small cell lung cancer: an update.” Molecular Cancer patent is extended or adjusted under 35 2008, 7.81. U.S.C. 154(b) by 0 days. * cited by examiner Primary Examiner — Gary BenZion (21) Appl. No.: 13/472,209 Assistant Examiner — Cynthia Wilder (74) Attorney, Agent, or Firm — Thomas C. Meyers; Brown (22) Filed: May 15, 2012 Rudnick LLP (57) ABSTRACT Primers having abasic regions or mismatches for amplifying (51) Int. C. sequences suspected of having methylation. Primers having CI2P 19/34 (2006.01) abasic regions or mismatches for amplifying sequences adja C7H 2L/04 (2006.01) cent to Suspected or known methylated sequences. Methods (52) U.S. Cl. .................................... 435/91.2:536/24.33 of using primers having abasic regions or mismatches for (58) Field of Classification Search ............... 435/24.33, identification of methylated sequences or sequences adjacent 435/91.2 to suspected or known methylation sequences. See application file for complete search history. 8 Claims, 8 Drawing Sheets U.S. Patent Feb. 19, 2013 Sheet 6 of 8 US 8,377,657 B1 TWIST qPCR using ForA4a and Rev1. Blue: 1.2uM primer Red: 1.OuM primer Pink: 600nM primer Light blue: 800nM primer Grey: 300nM primer Yellow water FIG 6 U.S. Patent Feb. 19, 2013 Sheet 8 of 8 US 8,377,657 B1 NID qPCR Primer concentration: 300nM-1.2uM FIG 8 US 8,377,657 B1 1. 2 PRMERS FOR ANALYZING METHYLATED using a single primerset. Aspects of the invention are accom SEQUENCES AND METHODS OF USE plished by using primers configured Such that they are able to THEREOF specifically hybridize to either a methylated or an unmethy lated site, e.g., a CpG island, of a template nucleic acid. FIELD OF THE INVENTION Aspects of the invention are also accomplished by using primers configured such that they are able to specifically The present invention generally relates to a primers con hybridize adjacent to a methylated or an unmethylated site, figured to specifically hybridize to either a methylated or an e.g., a CpG island, of a template nucleic acid. Accordingly, unmethylated CpG site of a template nucleic acid or adjacent there is no need to have different primer sequences that dis to a methylated or an unmethylated CpG site of a template tinguish between converted uracil sequences and uncon nucleic acid, and methods of use thereof. 10 Verted cytosine sequences. Thus, amplification reactions are performed on nucleic acid with a single set of primers, which BACKGROUND reduces costs and lowers assay complexity. One way that this is accomplished is by providing primers DNA methylation is an important regulator of gene expres that include an abasic region that interacts with either the sion and may play a role in the development and progression 15 methylated or the unmethylated CpG site of the template of a number of diseases, such as cancer. Methylation is typi nucleic acid. For example, a primer may contain one or more cally limited to cytosines located 5' to a guanine (i.e., CpG abasic regions corresponding to expected locations of methy sequences), however other forms of methylation are known. lation sites. Often the abasic region will be linked to a guanine Research suggests that genes with high levels of methylation moiety. The primer may contain any known abasic (spacer) in a promoter region are transcriptionally silent, which may molecule that is known in the art. Another way to accomplish allow unchecked cell proliferation. this goal is to provide primers including at least one mis When a promoter region has excessive methylation, the matched nucleotide that has similar annealing characteristics methylation is typically most prevalent in sequences having to both uracil and cytosine such that the primer hybridizes to CpG repeats, so called “CpG islands.” Undermethylation (hy either the methylated or the unmethylated CpG site of the pomethylation) has also been implicated in the development 25 template nucleic acid. Another way to accomplish this goal is and progression of cancer through different mechanisms. to provide primers that hybridize to sequences adjacent to Several methods have been developed to identify and quan methylated or the unmethylated CpG site(s) of the template tify methylation, especially in CpG sites, e.g., CpG islands, nucleic acid. The primer set allows for amplification of the that are implicated in silencing promoters. Those include entire site. Analysis of the amplification products gives infor sequencing methods in which genomic DNA is isolated and 30 mation about the methylation status of the sample. treated with bisulfite. Because methylated cytosines are not In some embodiments, the primers may include an adaptor affected by bisulfite treatment, the unmethylated Cs, e.g., sequence such that amplicons are produced with adaptors within a CpG site, are converted to uracil, while methylated already attached. In other embodiments, adaptors are Cs are not converted. After sequencing, comparison of the attached to the amplicons after the amplification reaction. The starting DNA and the bisulfate treated DNA indicates the 35 adaptor sequence may optionally include a homopolymer location of methylation sites. region, e.g., a poly-A region. In some embodiment, the primer Perhaps the most widely-used method of probing methy can specifically hybridize to either the methylated or the lation patterns is methylation specific PCR (MSP) which uses unmethylated CpG site of the template nucleic acid under two sets of primers for an amplification reaction. One primer conditions of high Stringency. In some instances, it is useful to set is complimentary to sequences whose Cs are converted to 40 use sequencing to analyze the amplification products. In these Us by bisulfite treatment, and the other primer set is compli embodiments it may be helpful to have a universal adaptor on mentary to non-converted Cs. Using these two separate the amplicons so that they can be hybridized to a universal primer sets, both the methylated and unmethylated DNA are primer on a solid Support for the sequencing reaction. amplified. Comparison of the amplification products gives Primers of the invention may be used in a number of appli insight as to the methylation in a given sequence. See Herman 45 cations where it is desirable to analyze methylation of a et al., “Methylation-specific PCR: A novel PCR assay for sequence, e.g., epigenetics or diagnosing a disease, e.g., can methylation status of CpG islands.” PN.A.S., vol. 93, p. 9821 cer. For example, a methylation pattern of a nucleic acid may 26 (1996), which is incorporated herein by reference in its be analyzed by obtaining template nucleic acid, contacting entirety. This technique can detect methylation changes as the template nucleic acid with an agent (e.g., bisulfite) that small as +0.1%. In addition to methylation of CpG islands, 50 modifies unmethylated cytosine, hybridizing a primer con many of the sequences Surrounding clinically relevant hyper figured such that it is able to specifically hybridize to either a methylated CpG islands can also be hypermethylated, and are methylated or an unmethylated CpG site of the template potential biomarkers. nucleic acid, amplifying the template nucleic acid, and ana A problem with MSP is that it requires the use of two lyzing a methylation pattern of the amplified nucleic acid. different primer sets, one for sequences containing methy 55 Any amplification or analysis method may be used in the lated Cs, and the other for sequences containing Us, which methods of the invention. For example, PCR amplification, were converted from unmethylated Cs by bisulfite treatment. direct sequencing, fluorescent probe hybridization, etc. In Using two different primer sets limits the application of MSP. Some instances, the template nucleic acid is amplified with In addition to the costs associated with producing and main PCR. In some instances, the amplified nucleic acid in ana taining two separate primer sets, the amplification process 60 lyzed by sequencing the nucleic acid. cannot be maximally efficient because of the need to operate in temperature regimes appropriate for both primer sets. BRIEF DESCRIPTION OF THE DRAWINGS SUMMARY FIG. 1 depicts a TWIST sequence with various regions of 65 potential methylation, and forward and reverse primers hav The invention provides compositions and methods for per ing abasic regions which can be used to amplify the TWIST forming methylation specific amplification of nucleic acids Sequence; US 8,377,657 B1 3 4 FIG. 2 depicts a NID sequence with various regions of sequences, e.g., CpG islands, and helps to suppress the potential methylation, and forward and reverse primers hav expression and mobility of transposable elements. Because ing abasic regions which can be used to amplify the NID 5-methylcytosine is chemically very similar to thymidine, Sequence; CpG sites are frequently mutated and become rare in the FIG.3 depicts a TWIST sequence with various regions of 5 genome, except at CpG islands where they remain unmethy potential methylation, and forward and reverse primers hav lated.
Load more

Recommended publications
  • Genomic and Expression Profiling of Chromosome 17 in Breast Cancer Reveals Complex Patterns of Alterations and Novel Candidate Genes
    [CANCER RESEARCH 64, 6453–6460, September 15, 2004] Genomic and Expression Profiling of Chromosome 17 in Breast Cancer Reveals Complex Patterns of Alterations and Novel Candidate Genes Be´atrice Orsetti,1 Me´lanie Nugoli,1 Nathalie Cervera,1 Laurence Lasorsa,1 Paul Chuchana,1 Lisa Ursule,1 Catherine Nguyen,2 Richard Redon,3 Stanislas du Manoir,3 Carmen Rodriguez,1 and Charles Theillet1 1Ge´notypes et Phe´notypes Tumoraux, EMI229 INSERM/Universite´ Montpellier I, Montpellier, France; 2ERM 206 INSERM/Universite´ Aix-Marseille 2, Parc Scientifique de Luminy, Marseille cedex, France; and 3IGBMC, U596 INSERM/Universite´Louis Pasteur, Parc d’Innovation, Illkirch cedex, France ABSTRACT 17q12-q21 corresponding to the amplification of ERBB2 and collinear genes, and a large region at 17q23 (5, 6). A number of new candidate Chromosome 17 is severely rearranged in breast cancer. Whereas the oncogenes have been identified, among which GRB7 and TOP2A at short arm undergoes frequent losses, the long arm harbors complex 17q21 or RP6SKB1, TBX2, PPM1D, and MUL at 17q23 have drawn combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array- most attention (6–10). Furthermore, DNA microarray studies have comparative genomic hybridization and of associated RNA expression revealed additional candidates, with some located outside current changes by cDNA arrays. We built a genomic array covering the entire regions of gains, thus suggesting the existence of additional amplicons chromosome at an average density of 1 clone per 0.5 Mb, and patterns of on 17q (8, 9). gains and losses were characterized in 30 breast cancer cell lines and 22 Our previous loss of heterozygosity mapping data pointed to the primary tumors.
    [Show full text]
  • Molecular Effects of Isoflavone Supplementation Human Intervention Studies and Quantitative Models for Risk Assessment
    Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis committee Promotors Prof. Dr Pieter van ‘t Veer Professor of Nutritional Epidemiology Wageningen University Prof. Dr Evert G. Schouten Emeritus Professor of Epidemiology and Prevention Wageningen University Co-promotors Dr Anouk Geelen Assistant professor, Division of Human Nutrition Wageningen University Dr Lydia A. Afman Assistant professor, Division of Human Nutrition Wageningen University Other members Prof. Dr Jaap Keijer, Wageningen University Dr Hubert P.J.M. Noteborn, Netherlands Food en Consumer Product Safety Authority Prof. Dr Yvonne T. van der Schouw, UMC Utrecht Dr Wendy L. Hall, King’s College London This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 20 June 2014 at 13.30 p.m. in the Aula. Vera van der Velpen Molecular effects of isoflavone supplementation: Human intervention studies and quantitative models for risk assessment 154 pages PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English ISBN: 978-94-6173-952-0 ABSTRact Background: Risk assessment can potentially be improved by closely linked experiments in the disciplines of epidemiology and toxicology.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • 1471-2105-8-217.Pdf
    BMC Bioinformatics BioMed Central Software Open Access GenMAPP 2: new features and resources for pathway analysis Nathan Salomonis1,2, Kristina Hanspers1, Alexander C Zambon1, Karen Vranizan1,3, Steven C Lawlor1, Kam D Dahlquist4, Scott W Doniger5, Josh Stuart6, Bruce R Conklin1,2,7,8 and Alexander R Pico*1 Address: 1Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158 USA, 2Pharmaceutical Sciences and Pharmacogenomics Graduate Program, University of California, 513 Parnassus Avenue, San Francisco, CA 94143, USA, 3Functional Genomics Laboratory, University of California, Berkeley, CA 94720 USA, 4Department of Biology, Loyola Marymount University, 1 LMU Drive, MS 8220, Los Angeles, CA 90045 USA, 5Computational Biology Graduate Program, Washington University School of Medicine, St. Louis, MO 63108 USA, 6Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064 USA, 7Department of Medicine, University of California, San Francisco, CA 94143 USA and 8Department of Molecular and Cellular Pharmacology, University of California, San Francisco, CA 94143 USA Email: Nathan Salomonis - [email protected]; Kristina Hanspers - [email protected]; Alexander C Zambon - [email protected]; Karen Vranizan - [email protected]; Steven C Lawlor - [email protected]; Kam D Dahlquist - [email protected]; Scott W Doniger - [email protected]; Josh Stuart - [email protected]; Bruce R Conklin - [email protected]; Alexander R Pico* - [email protected] * Corresponding author Published: 24 June 2007 Received: 16 November 2006 Accepted: 24 June 2007 BMC Bioinformatics 2007, 8:217 doi:10.1186/1471-2105-8-217 This article is available from: http://www.biomedcentral.com/1471-2105/8/217 © 2007 Salomonis et al; licensee BioMed Central Ltd.
    [Show full text]
  • A Graph-Theoretic Approach to Model Genomic Data and Identify Biological Modules Asscociated with Cancer Outcomes
    A Graph-Theoretic Approach to Model Genomic Data and Identify Biological Modules Asscociated with Cancer Outcomes Deanna Petrochilos A dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2013 Reading Committee: Neil Abernethy, Chair John Gennari, Ali Shojaie Program Authorized to Offer Degree: Biomedical Informatics and Health Education UMI Number: 3588836 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI 3588836 Published by ProQuest LLC (2013). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 ©Copyright 2013 Deanna Petrochilos University of Washington Abstract Using Graph-Based Methods to Integrate and Analyze Cancer Genomic Data Deanna Petrochilos Chair of the Supervisory Committee: Assistant Professor Neil Abernethy Biomedical Informatics and Health Education Studies of the genetic basis of complex disease present statistical and methodological challenges in the discovery of reliable and high-confidence genes that reveal biological phenomena underlying the etiology of disease or gene signatures prognostic of disease outcomes. This dissertation examines the capacity of graph-theoretical methods to model and analyze genomic information and thus facilitate using prior knowledge to create a more discrete and functionally relevant feature space.
    [Show full text]
  • Characterizing Genomic Duplication in Autism Spectrum Disorder by Edward James Higginbotham a Thesis Submitted in Conformity
    Characterizing Genomic Duplication in Autism Spectrum Disorder by Edward James Higginbotham A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Edward James Higginbotham 2020 i Abstract Characterizing Genomic Duplication in Autism Spectrum Disorder Edward James Higginbotham Master of Science Graduate Department of Molecular Genetics University of Toronto 2020 Duplication, the gain of additional copies of genomic material relative to its ancestral diploid state is yet to achieve full appreciation for its role in human traits and disease. Challenges include accurately genotyping, annotating, and characterizing the properties of duplications, and resolving duplication mechanisms. Whole genome sequencing, in principle, should enable accurate detection of duplications in a single experiment. This thesis makes use of the technology to catalogue disease relevant duplications in the genomes of 2,739 individuals with Autism Spectrum Disorder (ASD) who enrolled in the Autism Speaks MSSNG Project. Fine-mapping the breakpoint junctions of 259 ASD-relevant duplications identified 34 (13.1%) variants with complex genomic structures as well as tandem (193/259, 74.5%) and NAHR- mediated (6/259, 2.3%) duplications. As whole genome sequencing-based studies expand in scale and reach, a continued focus on generating high-quality, standardized duplication data will be prerequisite to addressing their associated biological mechanisms. ii Acknowledgements I thank Dr. Stephen Scherer for his leadership par excellence, his generosity, and for giving me a chance. I am grateful for his investment and the opportunities afforded me, from which I have learned and benefited. I would next thank Drs.
    [Show full text]
  • Endocrine System Local Gene Expression
    Copyright 2008 By Nathan G. Salomonis ii Acknowledgments Publication Reprints The text in chapter 2 of this dissertation contains a reprint of materials as it appears in: Salomonis N, Hanspers K, Zambon AC, Vranizan K, Lawlor SC, Dahlquist KD, Doniger SW, Stuart J, Conklin BR, Pico AR. GenMAPP 2: new features and resources for pathway analysis. BMC Bioinformatics. 2007 Jun 24;8:218. The co-authors listed in this publication co-wrote the manuscript (AP and KH) and provided critical feedback (see detailed contributions at the end of chapter 2). The text in chapter 3 of this dissertation contains a reprint of materials as it appears in: Salomonis N, Cotte N, Zambon AC, Pollard KS, Vranizan K, Doniger SW, Dolganov G, Conklin BR. Identifying genetic networks underlying myometrial transition to labor. Genome Biol. 2005;6(2):R12. Epub 2005 Jan 28. The co-authors listed in this publication developed the hierarchical clustering method (KP), co-designed the study (NC, AZ, BC), provided statistical guidance (KV), co- contributed to GenMAPP 2.0 (SD) and performed quantitative mRNA analyses (GD). The text of this dissertation contains a reproduction of a figure from: Yeo G, Holste D, Kreiman G, Burge CB. Variation in alternative splicing across human tissues. Genome Biol. 2004;5(10):R74. Epub 2004 Sep 13. The reproduction was taken without permission (chapter 1), figure 1.3. iii Personal Acknowledgments The achievements of this doctoral degree are to a large degree possible due to the contribution, feedback and support of many individuals. To all of you that helped, I am extremely grateful for your support.
    [Show full text]
  • An Integrated Data Analysis Approach to Characterize Genes Highly Expressed in Hepatocellular Carcinoma
    Oncogene (2005) 24, 3737–3747 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma Mohini A Patil1,6, Mei-Sze Chua2,6, Kuang-Hung Pan3,6, Richard Lin3, Chih-Jian Lih3, Siu-Tim Cheung4, Coral Ho1,RuiLi2, Sheung-Tat Fan4, Stanley N Cohen3, Xin Chen1,5 and Samuel So2 1Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143, USA; 2Department of Surgery and Asian Liver Center, Stanford University, Stanford, CA 94305, USA; 3Department of Genetics, Stanford University, Stanford, CA 94305, USA; 4Department of Surgery and Center for the Study of Liver Disease, University of Hong Kong, Hong Kong, China; 5Liver Center, University of California, San Francisco, CA 94143, USA Hepatocellular carcinoma (HCC) is one of the major cancer deaths worldwide (Parkin, 2001; Parkin et al., causes of cancer deaths worldwide. New diagnostic and 2001). Epidemiological and molecular genetic studies therapeutic options are needed for more effective and early have demonstrated that the development of HCC spans detection and treatment of this malignancy. We identified several decades, often starting with hepatitis B virus 703 genes that are highly expressed in HCC using DNA (HBV) or hepatitis C virus (HCV) infections. Chronic microarrays, and further characterized them in order to carriers of HBV or HCV are at much higher risk of uncover novel tumor markers, oncogenes, and therapeutic developing HCC, especially when infection has been targets for HCC. Using Gene Ontology annotations, genes accompanied by liver cirrhosis (El-Serag, H., 2001; El- with functions related to cell proliferation and cell cycle, Serag, H.B., 2002).
    [Show full text]
  • Gender-Specific Expression of Ubiquitin-Specific Peptidase 9 Modulates Tau Expression and Phosphorylation: Possible Implications for Tauopathies
    Mol Neurobiol DOI 10.1007/s12035-016-0299-z Gender-Specific Expression of Ubiquitin-Specific Peptidase 9 Modulates Tau Expression and Phosphorylation: Possible Implications for Tauopathies Sandra Köglsberger1 & Maria Lorena Cordero-Maldonado1 & Paul Antony1 & Julia Ilona Forster1 & Pierre Garcia 1 & Manuel Buttini1 & Alexander Crawford1 & Enrico Glaab1 Received: 22 May 2016 /Accepted: 14 November 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Public transcriptomic studies have shown that sev- literature, we derive a data-congruent model for a USP9-de- eral genes display pronounced gender differences in their ex- pendent regulatory mechanism modulating MAPT expression pression in the human brain, which may influence the mani- via BACH1 and SMAD4. Overall, the analyses suggest USP9 festations and risk for neuronal disorders. Here, we apply a may contribute to molecular gender differences observed in transcriptome-wide analysis to discover genes with gender- tauopathies and provide a new target for intervention strate- specific expression and significant alterations in public post- gies to modulate MAPT expression. mortem brain tissue from Alzheimer’s disease (AD) patients compared to controls. We identify the sex-linked ubiquitin- Keywords Transcriptomics .Tau .Tauopathies .Alzheimer’s specific peptidase 9 (USP9) as an outstanding candidate gene disease . Gender differences . Zebrafish with highly significant expression differences between the genders and male-specific underexpression in
    [Show full text]
  • Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes
    HIGHLIGHTED ARTICLE | INVESTIGATION Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes Riddhiman K. Garge,* Jon M. Laurent,*,† Aashiq H. Kachroo,‡,1 and Edward M. Marcotte*,1 *Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Texas 78712, †Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016, and ‡The Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montreal, H4B 1R6 Quebec, Canada ORCID IDs: 0000-0002-6774-0172 (R.K.G.); 0000-0001-6583-4741 (J.M.L.); 0000-0001-9770-778X (A.H.K.); 0000-0001-8808-180X (E.M.M.) ABSTRACT Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes, including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing 81% of human cytoskeletal genes across seven gene families for their ability to complement a growth defect induced by inactivation or deletion of the corresponding yeast ortholog. In five of seven families—all but a-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with deletion phenotypes of their interaction partners, we identify instances of human genes in the actin and septin families capable of carrying out essential functions, but failing to fully complement the cytoskeletal roles of their yeast orthologs, thus leading to abnormal cell morphologies.
    [Show full text]
  • Quantitative Trait Loci Mapping of Macrophage Atherogenic Phenotypes
    QUANTITATIVE TRAIT LOCI MAPPING OF MACROPHAGE ATHEROGENIC PHENOTYPES BRIAN RITCHEY Bachelor of Science Biochemistry John Carroll University May 2009 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY December 2017 We hereby approve this thesis/dissertation for Brian Ritchey Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies by ______________________________ Date: _________ Dissertation Chairperson, Johnathan D. Smith, PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, David J. Anderson, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Baochuan Guo, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Stanley L. Hazen, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Renliang Zhang, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Aimin Zhou, PhD Department of Chemistry, Cleveland State University Date of Defense: October 23, 2017 DEDICATION I dedicate this work to my entire family. In particular, my brother Greg Ritchey, and most especially my father Dr. Michael Ritchey, without whose support none of this work would be possible. I am forever grateful to you for your devotion to me and our family. You are an eternal inspiration that will fuel me for the remainder of my life. I am extraordinarily lucky to have grown up in the family I did, which I will never forget.
    [Show full text]
  • Datasheet: MCA4403Z Product Details
    Datasheet: MCA4403Z Description: MOUSE ANTI HUMAN TUBULIN GAMMA 2 CHAIN:Preservative Free Specificity: TUBULIN GAMMA 2 CHAIN Other names: TUBG2 Format: Preservative Free Product Type: Monoclonal Antibody Clone: 4F6 Isotype: IgG2a Quantity: 0.1 mg Product Details Applications This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit www.bio-rad-antibodies.com/protocols. Yes No Not Determined Suggested Dilution Flow Cytometry Immunohistology - Frozen Immunohistology - Paraffin ELISA Immunoprecipitation Western Blotting 0.1 - 10 ug/ml Immunofluorescence (1) 0.1 - 10 ug/ml Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls. (1) This product requires fixation of cells for 15 minutes. 4 % paraformaldehyde solution (NaOH in PBS) pH 7.4 is recommended for this purpose. Target Species Human Product Form Purified IgG - liquid Preparation Purified IgG prepared by affinity chromatography on Protein A Buffer Solution Phosphate buffered saline Preservative None present Stabilisers Approx. Protein IgG concentration 0.5 mg/ml Concentrations Immunogen Recombinant protein with a GST tag corresponding to amino acids 1 - 101 of human TUBG2. Page 1 of 3 External Database Links UniProt: Q9NRH3 Related reagents Entrez Gene: 27175 TUBG2 Related reagents Fusion Partners Spleen cells from BALB/c mice were fused with cells from the Sp2/0 myeloma cell line.
    [Show full text]