Transcriptional Regulation of the Human Alcohol

Total Page:16

File Type:pdf, Size:1020Kb

Transcriptional Regulation of the Human Alcohol TRANSCRIPTIONAL REGULATION OF THE HUMAN ALCOHOL DEHYDROGENASES AND ALCOHOLISM Sirisha Pochareddy Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University September 2010 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Howard J. Edenberg, Ph.D., Chair Maureen A. Harrington, Ph.D. Doctoral Committee David G. Skalnik, Ph.D. Ann Roman, Ph.D. July 30, 2010 ii This work is dedicated to my parents and my brother for their unwavering support and unconditional love. iii ACKNOWLEDGEMENTS I would like to sincerely thank my mentor Dr. Howard Edenberg, for his guidance, support throughout the five years of my research in his lab. It has been an amazing learning experience working with him and I am confident this training will help all through my research career. I would like to thank members of my research committee, Dr. Maureen Harrington, Dr. David Skalnik and Dr. Ann Roman. I am grateful to them for their guidance, encouraging comments, time and effort. I greatly appreciate Dr. Harrington’s questions during the committee meeting that helped me think broadly about my area of research. I am very thankful to Dr. Skalnik for reading through my manuscript and giving his valuable comments. My special thanks to Dr. Ann Roman for staying on my committee even after her retirement. I am also thankful to Dr. Jeanette McClintick for her patience in answering my never ending list of questions about the microarray analysis. She also had been a great support during the tough times in the lab. I would like to thank her making an effort to remember birthdays of all lab members and baking her awesome brownies. I would like to thank other lab members, Ron Jerome, Jun Wang and Sowmya Jairam. It was a great pleasure to know Ron during the last year of my stay. He made the toughest years of Ph.D. less stressful and more fun. Jun was always helpful in the lab. I am also thankful to Sowmya for sharing her ideas with me and helping me think more about ADH transcriptional regulation. I would also like to thank Dr. Xiaoling Xuei and Dr. Yunlong Liu for all their help. iv I would like to thank my best friends, Dr. Sirisha Asuri and Dr. Raji Muthukrishnan for their beautiful, unconditional friendship. I am also thankful to my other friends Sulo, Aditi, Heather, and Chandra for all the fun. Finally, I would like to thank my family members. My mom Prabhavathy and my dad P.S. Reddy have been there for me always, supporting all my decisions. They have been with me through the highs and the lows and always made me believe that everything is going to be fine. My dream of doing research and getting a Ph.D. would not have been possible without their strong emotional support. Another pillar of support in my life is my brother Subhash. He is my guide, teacher, friend, brother and has been a great source of strength in the most difficult times. Anna, thank you so much for everything. I would also like to thank my sister-in-law, Jhansi for being a sister I never had and a great friend. Lastly, I would like to thank cute little ones - my nephew Arjun, my niece Megha, Nishant, Niha and Charan, for lifting my spirits with their innocent smiles. v ABSTRACT Sirisha Pochareddy TRANSCRIPTIONAL REGULATION OF THE HUMAN ALCOHOL DEHYDROGENASES AND ALCOHOLISM Alcohol dehydrogenase (ADH) genes encode proteins that metabolize ethanol to acetaldehyde. Humans have seven ADH genes in a cluster. The hypothesis of this study was that by controlling the levels of ADH enzymes, cis- regulatory regions could affect the risk for alcoholism. The goal was thus to identify distal regulatory regions of ADHs. To achieve this, sequence conservation across 220 kb of the ADH cluster was examined. An enhancer (4E) was identified upstream of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell specific, as no enhancer activity was detected in a human lung cell line, H1299. The enhancer activity was located in a 565 bp region (4E3). Four FOXA and one HNF-1A protein binding sites were shown to be functional in the 4E3 region. To test if this region could affect the risk for alcoholism, the effect of variations in 4E3 on enhancer activity was tested. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold. A third variation had a small but significant effect. The effect of variations in the ADH1B proximal promoter was also tested. At SNP rs1229982, the C allele had 30% lower activity than the A allele. vi In addition to studying the regulatory regions of ADH genes, the effects of alcohol on liver-derived cells (HepG2) were also explored. Liver is the primary site of alcohol metabolism, and is highly vulnerable to injuries due to chronic alcohol abuse. To identify the effects of long term ethanol exposure on global gene expression and alternative splicing, HepG2 cells were cultured in 75 mM ethanol for nine days. Global gene expression changes and alternative splicing were measured using Affymetrix GeneChip® Human Exon 1.0 ST Arrays. At the level of gene expression, genes involved in stress response pathways, metabolic pathways (including carbohydrate and lipid metabolism) and chromatin regulation were affected. Alcohol effects were also observed on alternative transcript isoforms of some genes. Howard J. Edenberg, Ph.D. Committee Chair. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................. xiii ABBREVIATIONS ............................................................................................... xiv I. INTRODUCTION ............................................................................................... 1 1. Alcohol dehydrogenases .............................................................................. 1 2. Human ADH cluster ...................................................................................... 5 3. Additional pathways of alcohol metabolism .................................................. 6 4. Alcoholism .................................................................................................... 7 5. ADHs and alcoholism ................................................................................... 9 6. Transcriptional regulation of ADHs ............................................................. 11 7. Identification of cis-regulatory regions ........................................................ 17 8. Transcription factors ................................................................................... 18 8.a. FoxA family ......................................................................................... 19 8.b. HNF-1A ............................................................................................... 20 9. Alcohol and the liver ................................................................................... 21 10. Alternative transcript isoforms and diseases ............................................ 24 11. Global transcriptional profiling .................................................................. 27 12. Research objectives ................................................................................. 32 viii II. MATERIALS AND METHODS ........................................................................ 34 1. Identification of putative distal regulatory elements .................................... 34 2. Cloning of test fragments ............................................................................ 34 3. Transient transfections and reporter gene assays ...................................... 38 4. Electrophoretic mobility shift assays (EMSA) ............................................. 40 5. Site directed mutagenesis .......................................................................... 42 6. Generation of the 4E haplotypes ................................................................ 42 7. Long-term treatment of HepG2 cells with ethanol....................................... 44 8. RNA extraction, labeling and hybridization ................................................. 44 9. Exon array data analysis ............................................................................ 45 10. Validation of differential gene expression by qRT-PCR ............................ 51 11. Validation of alternative splicing by qRT-PCR .......................................... 52 III. RESULTS ....................................................................................................... 54 1. Identification of an enhancer in the ADH cluster ......................................... 54 2. Characterization of the enhancer element 4E ............................................ 58 2.a. Effect of 4E on heterologous promoters .............................................. 58 2.b. Function of 4E in non-hepatoma cells ................................................. 58 2.c. Localization of sequences required for 4E enhancer activity ............... 59 2.d. Identification of potential protein binding sites in 4E ........................... 61 2.e. Effect of mutations on enhancer activity.............................................. 66 ix 3. Effects of regulatory variations on gene expression
Recommended publications
  • Proteomic Analyses Reveal a Role of Cytoplasmic Droplets As an Energy Source During Sperm Epididymal Maturation
    Proteomic analyses reveal a role of cytoplasmic droplets as an energy source during sperm epididymal maturation Shuiqiao Yuana,b, Huili Zhenga, Zhihong Zhengb, Wei Yana,1 aDepartment of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557; and bDepartment of Laboratory Animal Medicine, China Medical University, Shenyang, 110001, China Corresponding author. Email: [email protected] Supplemental Information contains one Figure (Figure S1), three Tables (Tables S1-S3) and two Videos (Videos S1 and S2) files. Figure S1. Scanning electron microscopic images of purified murine cytoplasmic droplets. Arrows point to indentations resembling the resealed defects at the detaching points when CDs come off the sperm flagella. Scale bar = 1µm Table S1 Mass spectrometry-based identifiaction of proteins highly enriched in murine cytoplasmic droplets. # MS/MS View:Identified Proteins (105) Accession Number Molecular Weight Protein Grouping Ambiguity Dot_1_1 Dot_2_1 Dot_3_1 Dot_4_1Dot_5_1 Dot_1_2 Dot_2_2 Dot_3_2 Dot_4_2 Dot_5_2 1 IPI:IPI00467457.3 Tax_Id=10090 Gene_Symbol=Ldhc L-lactate dehydrogenase C chain IPI00467457 36 kDa TRUE 91% 100% 100% 100% 100% 100% 100% 100% 100% 2 IPI:IPI00473320.2 Tax_Id=10090 Gene_Symbol=Actb Putative uncharacterized protein IPI00473320 42 kDa TRUE 75% 100% 100% 100% 100% 89% 76% 100% 100% 100% 3 IPI:IPI00224181.7 Tax_Id=10090 Gene_Symbol=Akr1b7 Aldose reductase-related protein 1 IPI00224181 36 kDa TRUE 100% 100% 76% 100% 100% 4 IPI:IPI00228633.7 Tax_Id=10090 Gene_Symbol=Gpi1 Glucose-6-phosphate
    [Show full text]
  • Cytoplasmic Activation-Induced Cytidine Deaminase (AID) Exists in Stoichiometric Complex with Translation Elongation Factor 1Α (Eef1a)
    Cytoplasmic activation-induced cytidine deaminase (AID) exists in stoichiometric complex with translation elongation factor 1α (eEF1A) Julien Häsler, Cristina Rada, and Michael S. Neuberger1 Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom Edited by Frederick W. Alt, Howard Hughes Medical Institute, Harvard Medical School, Children’s Hospital Immune Disease Institute, Boston, MA, and approved October 12, 2011 (received for review April 27, 2011) Activation-induced cytidine deaminase (AID) is a B lymphocyte- results reveal that endogenous cytoplasmic AID partakes in a specific DNA deaminase that acts on the Ig loci to trigger antibody complex containing stoichiometric quantities of translation elon- gene diversification. Most AID, however, is retained in the cyto- gation factor 1α (eEF1A), with this association likely implicated in plasm and its nuclear abundance is carefully regulated because the regulation of AID’s intracellular trafficking. off-target action of AID leads to cancer. The nature of the cytosolic AID complex and the mechanisms regulating its release from the Results cytoplasm and import into the nucleus remain unknown. Here, we Flag-Tagging the Endogenous AID Locus in DT40 Cells. We generated show that cytosolic AID in DT40 B cells is part of an 11S complex derivatives of the DT40 B-cell line in which the endogenous AID and, using an endogenously tagged AID protein to avoid overex- locus was modified so as to incorporate a single Flag tag at the pression artifacts, that it is bound in good stoichiometry to the AID N terminus. To allow targeting of both alleles, one targeting translation elongation factor 1 alpha (eEF1A).
    [Show full text]
  • The Title of the Article
    Mechanism-Anchored Profiling Derived from Epigenetic Networks Predicts Outcome in Acute Lymphoblastic Leukemia Xinan Yang, PhD1, Yong Huang, MD1, James L Chen, MD1, Jianming Xie, MSc2, Xiao Sun, PhD2, Yves A Lussier, MD1,3,4§ 1Center for Biomedical Informatics and Section of Genetic Medicine, Department of Medicine, The University of Chicago, Chicago, IL 60637 USA 2State Key Laboratory of Bioelectronics, Southeast University, 210096 Nanjing, P.R.China 3The University of Chicago Cancer Research Center, and The Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL 60637 USA 4The Institute for Genomics and Systems Biology, and the Computational Institute, The University of Chicago, Chicago, IL 60637 USA §Corresponding author Email addresses: XY: [email protected] YH: [email protected] JC: [email protected] JX: [email protected] XS: [email protected] YL: [email protected] - 1 - Abstract Background Current outcome predictors based on “molecular profiling” rely on gene lists selected without consideration for their molecular mechanisms. This study was designed to demonstrate that we could learn about genes related to a specific mechanism and further use this knowledge to predict outcome in patients – a paradigm shift towards accurate “mechanism-anchored profiling”. We propose a novel algorithm, PGnet, which predicts a tripartite mechanism-anchored network associated to epigenetic regulation consisting of phenotypes, genes and mechanisms. Genes termed as GEMs in this network meet all of the following criteria: (i) they are co-expressed with genes known to be involved in the biological mechanism of interest, (ii) they are also differentially expressed between distinct phenotypes relevant to the study, and (iii) as a biomodule, genes correlate with both the mechanism and the phenotype.
    [Show full text]
  • Genotyping for Response to Physical Training
    Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2019 Genotyping for Response to Physical Training Stacy Simmons Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Molecular Biology Commons Repository Citation Simmons, Stacy, "Genotyping for Response to Physical Training" (2019). Browse all Theses and Dissertations. 2109. https://corescholar.libraries.wright.edu/etd_all/2109 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. GENOTYPING FOR RESPONSE TO PHYSICAL TRAINING A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By STACY SIMMONS B.S., Wright State University, 2014 _________________________________________________________ 2019 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL July 29, 2019 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Stacy Simmons ENTITLED Genotyping for Response to Physical Training BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ___________________________________ Michael Markey, Ph.D. Thesis Director ____________________________________ Madhavi P. Kadakia, Ph.D. Committee on Chair, Department of Biochemistry Final Examination and
    [Show full text]
  • Functional Annotation of Exon Skipping Event in Human Pora Kim1,*,†, Mengyuan Yang1,†,Keyiya2, Weiling Zhao1 and Xiaobo Zhou1,3,4,*
    D896–D907 Nucleic Acids Research, 2020, Vol. 48, Database issue Published online 23 October 2019 doi: 10.1093/nar/gkz917 ExonSkipDB: functional annotation of exon skipping event in human Pora Kim1,*,†, Mengyuan Yang1,†,KeYiya2, Weiling Zhao1 and Xiaobo Zhou1,3,4,* 1School of Biomedical Informatics, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA, 2College of Electronics and Information Engineering, Tongji University, Shanghai, China, 3McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA and 4School of Dentistry, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA Received August 13, 2019; Revised September 21, 2019; Editorial Decision October 03, 2019; Accepted October 03, 2019 ABSTRACT been used as therapeutic targets (3–8). For example, MET has lost the binding site of E3 ubiquitin ligase CBL through Exon skipping (ES) is reported to be the most com- exon 14 skipping event (9), resulting in an enhanced expres- mon alternative splicing event due to loss of func- sion level of MET. MET amplification drives the prolifera- tional domains/sites or shifting of the open read- tion of tumor cells. Multiple tyrosine kinase inhibitors, such ing frame (ORF), leading to a variety of human dis- as crizotinib, cabozantinib and capmatinib, have been used eases and considered therapeutic targets. To date, to treat patients with MET exon 14 skipping (10). Another systematic and intensive annotations of ES events example is the dystrophin gene (DMD) in Duchenne mus- based on the skipped exon units in cancer and cular dystrophy (DMD), a progressive neuromuscular dis- normal tissues are not available.
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Accumulation of PNPLA3 on Lipid Droplets Is the Basis of Associated Hepatic Steatosis
    Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis Soumik BasuRaya, Yang Wanga, Eriks Smagrisa, Jonathan C. Cohenb,1, and Helen H. Hobbsa,b,c,1 aDepartment of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; and cHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390 Contributed by Helen H. Hobbs, March 19, 2019 (sent for review February 4, 2019; reviewed by Edward A. Fisher and Rudi Zechner) Fatty liver disease (FLD) is a disorder in which accumulation of causes steatosis whereas overexpression of the wild-type (WT) triglycerides (TGs) in the liver can lead to inflammation, fibrosis, and protein does not (15). In KI mice that express PNPLA3(148M) cirrhosis. Previously, we identified a variant (I148M) in patatin-like or PNPLA3(47A), the levels of PNPLA3 on hepatic lipid drop- phospholipase domain-containing protein 3 (PNPLA3) that is strongly lets (LDs) are ∼40-fold higher than those in WT mice, despite associated with FLD, but the mechanistic basis for the association similar levels of PNPLA3 mRNA in the two lines (16). A similar remains elusive. Although PNPLA3 has TG hydrolase activity in vitro, accumulation of PNPLA3 protein was observed in transgenic inactivation or overexpression of the WT protein in mice does not mice expressing human PNPLA3(148M) compared with mice cause steatosis. In contrast, expression of two catalytically defective expressing the WT transgene (15). The massive increase in forms of PNPLA3 (I148M or S47A) in sucrose-fed mice causes accumu- PNPLA3(148M and 47A) levels appears to be due to decreased lation of both PNPLA3 and TGs on hepatic lipid droplets (LDs).
    [Show full text]
  • An Overview of the Role of Hdacs in Cancer Immunotherapy
    International Journal of Molecular Sciences Review Immunoepigenetics Combination Therapies: An Overview of the Role of HDACs in Cancer Immunotherapy Debarati Banik, Sara Moufarrij and Alejandro Villagra * Department of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, The George Washington University, 800 22nd St NW, Suite 8880, Washington, DC 20052, USA; [email protected] (D.B.); [email protected] (S.M.) * Correspondence: [email protected]; Tel.: +(202)-994-9547 Received: 22 March 2019; Accepted: 28 April 2019; Published: 7 May 2019 Abstract: Long-standing efforts to identify the multifaceted roles of histone deacetylase inhibitors (HDACis) have positioned these agents as promising drug candidates in combatting cancer, autoimmune, neurodegenerative, and infectious diseases. The same has also encouraged the evaluation of multiple HDACi candidates in preclinical studies in cancer and other diseases as well as the FDA-approval towards clinical use for specific agents. In this review, we have discussed how the efficacy of immunotherapy can be leveraged by combining it with HDACis. We have also included a brief overview of the classification of HDACis as well as their various roles in physiological and pathophysiological scenarios to target key cellular processes promoting the initiation, establishment, and progression of cancer. Given the critical role of the tumor microenvironment (TME) towards the outcome of anticancer therapies, we have also discussed the effect of HDACis on different components of the TME. We then have gradually progressed into examples of specific pan-HDACis, class I HDACi, and selective HDACis that either have been incorporated into clinical trials or show promising preclinical effects for future consideration.
    [Show full text]
  • Katalog 2015 Cover Paul Lin *Hinweis Förderung.Indd
    Product List 2015 WE LIVE SERVICE Certificates quartett owns two productions sites that are certified according to EN ISO 9001:2008 Quality management systems - Requirements EN ISO 13485:2012 + AC:2012 Medical devices - Quality management systems - Requirements for regulatory purposes GMP Conformity Our quality management guarantees products of highest quality! 2 Foreword to the quartett product list 2015 quartett Immunodiagnostika, Biotechnologie + Kosmetik Vertriebs GmbH welcomes you as one of our new business partners as well as all of our previous loyal clients. You are now member of quartett´s worldwide customers. First of all we would like to introduce ourselves to you. Founded as a family-run company in 1986, quartett ensures for more than a quarter of a century consistent quality of products. Service and support of our valued customers are our daily businesses. And we will continue! In the end 80´s quartett offered radioimmunoassay and enzyme immunoassay kits from different manufacturers in the USA. In the beginning 90´s the company changed its strategy from offering products for routine diagnostic to the increasing field of research and development. Setting up a production plant in 1997 and a second one in 2011 supported this decision. The company specialized its product profile in the field of manufacturing synthetic peptides for antibody production, peptides such as protease inhibitors, biochemical reagents and products for histology, cytology and immunohistology. All products are exclusively manufactured in Germany without outsourcing any production step. Nowadays, we expand into all other diagnostic and research fields and supply our customers in universities, government institutes, pharmaceutical and biotechnological companies, hospitals, and private doctor offices.
    [Show full text]
  • Table 2. Significant
    Table 2. Significant (Q < 0.05 and |d | > 0.5) transcripts from the meta-analysis Gene Chr Mb Gene Name Affy ProbeSet cDNA_IDs d HAP/LAP d HAP/LAP d d IS Average d Ztest P values Q-value Symbol ID (study #5) 1 2 STS B2m 2 122 beta-2 microglobulin 1452428_a_at AI848245 1.75334941 4 3.2 4 3.2316485 1.07398E-09 5.69E-08 Man2b1 8 84.4 mannosidase 2, alpha B1 1416340_a_at H4049B01 3.75722111 3.87309653 2.1 1.6 2.84852656 5.32443E-07 1.58E-05 1110032A03Rik 9 50.9 RIKEN cDNA 1110032A03 gene 1417211_a_at H4035E05 4 1.66015788 4 1.7 2.82772795 2.94266E-05 0.000527 NA 9 48.5 --- 1456111_at 3.43701477 1.85785922 4 2 2.8237185 9.97969E-08 3.48E-06 Scn4b 9 45.3 Sodium channel, type IV, beta 1434008_at AI844796 3.79536664 1.63774235 3.3 2.3 2.75319499 1.48057E-08 6.21E-07 polypeptide Gadd45gip1 8 84.1 RIKEN cDNA 2310040G17 gene 1417619_at 4 3.38875643 1.4 2 2.69163229 8.84279E-06 0.0001904 BC056474 15 12.1 Mus musculus cDNA clone 1424117_at H3030A06 3.95752801 2.42838452 1.9 2.2 2.62132809 1.3344E-08 5.66E-07 MGC:67360 IMAGE:6823629, complete cds NA 4 153 guanine nucleotide binding protein, 1454696_at -3.46081884 -4 -1.3 -1.6 -2.6026947 8.58458E-05 0.0012617 beta 1 Gnb1 4 153 guanine nucleotide binding protein, 1417432_a_at H3094D02 -3.13334396 -4 -1.6 -1.7 -2.5946297 1.04542E-05 0.0002202 beta 1 Gadd45gip1 8 84.1 RAD23a homolog (S.
    [Show full text]
  • Supplementary Information Integrative Analyses of Splicing in the Aging Brain: Role in Susceptibility to Alzheimer’S Disease
    Supplementary Information Integrative analyses of splicing in the aging brain: role in susceptibility to Alzheimer’s Disease Contents 1. Supplementary Notes 1.1. Religious Orders Study and Memory and Aging Project 1.2. Mount Sinai Brain Bank Alzheimer’s Disease 1.3. CommonMind Consortium 1.4. Data Availability 2. Supplementary Tables 3. Supplementary Figures Note: Supplementary Tables are provided as separate Excel files. 1. Supplementary Notes 1.1. Religious Orders Study and Memory and Aging Project Gene expression data1. Gene expression data were generated using RNA- sequencing from Dorsolateral Prefrontal Cortex (DLPFC) of 540 individuals, at an average sequence depth of 90M reads. Detailed description of data generation and processing was previously described2 (Mostafavi, Gaiteri et al., under review). Samples were submitted to the Broad Institute’s Genomics Platform for transcriptome analysis following the dUTP protocol with Poly(A) selection developed by Levin and colleagues3. All samples were chosen to pass two initial quality filters: RNA integrity (RIN) score >5 and quantity threshold of 5 ug (and were selected from a larger set of 724 samples). Sequencing was performed on the Illumina HiSeq with 101bp paired-end reads and achieved coverage of 150M reads of the first 12 samples. These 12 samples will serve as a deep coverage reference and included 2 males and 2 females of nonimpaired, mild cognitive impaired, and Alzheimer's cases. The remaining samples were sequenced with target coverage of 50M reads; the mean coverage for the samples passing QC is 95 million reads (median 90 million reads). The libraries were constructed and pooled according to the RIN scores such that similar RIN scores would be pooled together.
    [Show full text]
  • Agency Response Letter GRAS Notice No. GRN 000675
    . Janet Oesterling Novozymes North America, Inc. 77 Perry Chapel Church Road Franklinton, NC 27525 Re: GRAS Notice No. GRN 000675 Dear Ms. Oesterling: The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000675. We received the notice, dated October 10, 2016, that you submitted in accordance with the agency’s proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal) on July 17, 2016, and filed it on October 14, 2016. We received amendments containing clarifying information on February, 22, 2017 and March 09, 2017. FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000675 was pending on the effective date of the GRAS final rule, we requested some additional information consistent with the format and requirements of the final rule. We received an amendment responding to this request on October 24, 2016. The subject of the notice is xylanase enzyme preparation produced by Trichoderma reesei carrying a xylanase gene from Talaromyces leycettanus (xylanase enzyme preparation). The notice informs us of the view of Novozymes North America, Inc. (Novozymes) that xylanase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme at levels up to 48.33 mg Total Organic Solids (TOS) per kg raw material in brewing, cereal beverage processing, baking, and processing of cereal grains such as corn, wheat, barley, and oats. Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes the chemical reaction as well as substances used as stabilizers, preservatives, or diluents.
    [Show full text]