DOCSLIB.ORG

Mammalian Germ Cells Are Determined After PGC Colonization of the Nascent Gonad

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mammalian Germ Cells Are Determined After PGC Colonization of the Nascent Gonad Mammalian germ cells are determined after PGC colonization of the nascent gonad Peter K. Nichollsa, Hubert Schorlea,b, Sahin Naqvia,c, Yueh-Chiang Hua,d,e, Yuting Fana,f, Michelle A. Carmella, Ina Dobrinskig, Adrienne L. Watsonh, Daniel F. Carlsonh, Scott C. Fahrenkrugh, and David C. Pagea,c,i,1 aWhitehead Institute, Cambridge, MA 02142; bDepartment of Developmental Pathology, Institute of Pathology, University of Bonn Medical School, 53127 Bonn, Germany; cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; dDivision of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; eDepartment of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45267; fReproductive Medicine Center, Sixth Affiliated Hospital, Sun Yat-sen University, 510655 Guangzhou, China; gDepartment of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; hRecombinetics, Inc., Saint Paul, MN 55104; and iHoward Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142 Contributed by David C. Page, October 15, 2019 (sent for review June 28, 2019; reviewed by Katherine L. Nathanson and Dustin L. Updike) Mammalian primordial germ cells (PGCs) are induced in the embry- transplanted to ectopic sites (8) and give rise to pluripotent cell onic epiblast, before migrating to the nascent gonads. In fish, lines in culture (9–11). It has also been suggested that pre- frogs, and birds, the germline segregates even earlier, through the sumptive PGCs (labeled genetically by Prdm1-Cre) in the pos- action of maternally inherited germ plasm. Across vertebrates, terior region of the embryo during allantoic elongation may migrating PGCs retain a broad developmental potential, regardless contribute to nongametogenic lineages (12, 13). Taken together, of whether they were induced or maternally segregated. In these observations suggest that migratory PGCs of vertebrates mammals, this potential is indicated by expression of pluripotency retain a broad developmental potential, regardless of their mode factors, and the ability to generate teratomas and pluripotent cell of segregation. That is, migratory PGCs, while clearly cells of the lines. How the germline loses this developmental potential re- germline (the entire lineage from zygote to gamete), may not yet mains unknown. Our genome-wide analyses of embryonic human be germ cells, which, by definition, are committed to producing and mouse germlines reveal a conserved transcriptional program, gametes and no other cell types (14). initiated in PGCs after gonadal colonization, that differentiates To better understand germline commitment in mammals, we germ cells from their germline precursors and from somatic line- DEVELOPMENTAL BIOLOGY ages. Through genetic studies in mice and pigs, we demonstrate examine the transition that occurs as PGCs invade the nascent that one such gonad-induced factor, the RNA-binding protein gonads. We find a transcriptional program, initiated in human and DAZL, is necessary in vivo to restrict the developmental potential mouse PGCs after colonization of the gonad, that distinguishes of the germline; DAZL’s absence prolongs expression of a Nanog germ cells from their migratory germline precursors, and from pluripotency reporter, facilitates derivation of pluripotent cell lines, soma. Through genetic studies, we demonstrate that this program and causes spontaneous gonadal teratomas. Based on these obser- is necessary for germ cell commitment in mammals. In embryonic vations in humans, mice, and pigs, we propose that germ cells are mice deficient in one factor induced at PGC colonization—the determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis— Significance after organogenesis has begun—in the common ancestor of all vertebrates, as in modern mammals, where this transition is in- In mammals, the germline is set aside early in development for duced by somatic cells of the gonad. We suggest that failure of the later production of the gametes, either eggs or sperm. It this process of germ cell determination likely accounts for the remains unknown when, and how, the precursor cells (termed origin of human testis cancer. primordial germ cells [PGCs]) become committed to produce only gametes, and no other cell type. We identify an embryonic germ cell | commitment | teratoma | pluripotency | Dazl transition occurring late in development, after PGCs colonize the nascent gonad, that serves to commit these cells to produce uring embryogenesis, cells segregate into germline and so- only gametes. Our findings have broad implications for the Dmatic lineages. In mammals, this split is first evident around origin of germ cell tumors in humans, and for the stepwise the time of gastrulation, when intercellular signaling induces the commitment of the germline in mammals and other vertebrates. formation of primordial germ cells (PGCs) (1, 2). Comparative studies reveal that an inductive method of germline segregation Author contributions: P.K.N. and D.C.P. conceptualized the project; P.K.N., H.S., S.N., I.D., likely existed in the common ancestor of all vertebrates (3). How- A.L.W., D.F.C., S.C.F., and D.C.P. designed research; P.K.N., H.S., S.N., Y.-C.H., Y.F., M.A.C., I.D., A.L.W., and D.F.C. performed research; P.K.N., Y.-C.H., M.A.C., I.D., A.L.W., D.F.C., and ever, some vertebrates, such as fish, frogs, and birds, have acquired S.C.F. contributed new reagents/analytic tools; P.K.N., H.S., S.N., Y.-C.H., Y.F., I.D., A.L.W., a different approach to germline segregation. It occurs much earlier and D.F.C. analyzed data; and P.K.N. and D.C.P. wrote the paper. in these species—during the first cell divisions of the zygote— Reviewers: K.L.N., Raymond and Ruth Perelman School of Medicine at the University of through the action of maternally supplied RNAs known as germ Pennsylvania; and D.L.U., Mount Desert Island Biological Laboratory. plasm (4). Competing interest statement: A.L.W., D.F.C., and S.C.F. are employees and shareholders Despite these different strategies for PGC formation, emerg- of Recombinetics Inc. I.D. is a member of the scientific advisory board of Recombinetics ing evidence suggests that migratory PGCs of nonmammalian Inc. The remaining authors have declared that no competing interests exist. vertebrates remain developmentally uncommitted to gametogen- Published under the PNAS license. esis, retaining the capacity for somatic differentiation. In frogs, Data deposition: Data generated using the Mouse Diversity Genotyping Array (Thermo Fisher Scientific) for the SNP genotyping of 129S2 and 129S4 substrains have been de- PGCs arising via germ plasm readily differentiate into somatic posited at the Gene Expression Omnibus under accession no. GSE87771. Data generated cells when transplanted into host embryos (5). Similarly, in fish, from control and Dazl-deficient germ line cells at E10.3 and E11.5 have been deposited mismigrated PGCs readily adopt somatic fates if depleted of Dnd1 under the Sequence Read Archive BioProject accession no. PRJNA434733. (6). In salamanders, where PGCs arise through inductive processes, 1To whom correspondence may be addressed. Email: [email protected]. irreversible commitment of the germline occurs late in develop- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ment, long after gastrulation is complete and somatic lineages are doi:10.1073/pnas.1910733116/-/DCSupplemental. established (7). In mammals, migratory PGCs can form teratomas if www.pnas.org/cgi/doi/10.1073/pnas.1910733116 PNAS Latest Articles | 1of11 Downloaded by guest on September 30, 2021 RNA-binding protein Dazl—the germline remains developmentally embryonic stem [ES] cells) (17–19) (SI Appendix, Fig. S3). uncommitted, retaining expression of a network of pluripotency [Pluripotency factors are similarly expressed in migratory PGCs factors, the capacity for pluripotent cell line derivation, and the of fish and birds (20, 21).] Further, in humans and mice of both potential to form gonadal teratomas in mice and pigs. sexes, these pluripotency factors are markedly down-regulated after PGC colonization and induction of the germ cell-defining Results program of gene expression (SI Appendix,Fig.S3). Might activa- Germ Cell Determinants of Nonmammalian Vertebrates Are Expressed tion of this program be necessary to down-regulate pluripotency in Mice and Humans upon PGC Colonization of the Nascent Gonads. We factors and restrict the developmental potential of the germline? searched for changes in gene expression that accompany PGC colonization of gonads in mammals. To this end, we reanalyzed Expression of Pluripotency Factors, and the Capacity for Deriving published transcriptomes of migratory and gonadal germline cells Pluripotent Cell Lines, Are Prolonged in Dazl-Deficient Mice. We from mouse (15) and human (16) embryos. Our analyses in mice considered whether the program of gene expression induced identified 74 genes whose expression increased robustly after after PGCs colonize the gonads functions in germ cell commit- PGCs colonized the gonads (fold change of >4 [presented as log2 ment. Evidence from a range of vertebrates suggests that DAZL, transformed] from E9.5 to E11.5, false discovery rate (FDR) < encoding an RNA-binding protein, might contribute to this 0.05; Fig. 1A and Datasets S1 and S2). To determine whether this function. For example,
Load more

Recommended publications
  • Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes
    G C A T T A C G G C A T genes Article Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes Filip Pajpach , Linda Shearwin-Whyatt and Frank Grützner * School of Biological Sciences, The University of Adelaide, Adelaide, SA 5005, Australia; fi[email protected] (F.P.); [email protected] (L.S.-W.) * Correspondence: [email protected] Abstract: Chromosome segregation at mitosis and meiosis is a highly dynamic and tightly regulated process that involves a large number of components. Due to the fundamental nature of chromosome segregation, many genes involved in this process are evolutionarily highly conserved, but duplica- tions and functional diversification has occurred in various lineages. In order to better understand the evolution of genes involved in chromosome segregation in mammals, we analyzed some of the key components in the basal mammalian lineage of egg-laying mammals. The chromosome passenger complex is a multiprotein complex central to chromosome segregation during both mitosis and meio- sis. It consists of survivin, borealin, inner centromere protein, and Aurora kinase B or C. We confirm the absence of Aurora kinase C in marsupials and show its absence in both platypus and echidna, which supports the current model of the evolution of Aurora kinases. High expression of AURKBC, an ancestor of AURKB and AURKC present in monotremes, suggests that this gene is performing all necessary meiotic functions in monotremes. Other genes of the chromosome passenger complex complex are present and conserved in monotremes, suggesting that their function has been preserved Citation: Pajpach, F.; in mammals.
    [Show full text]
  • Lecture9'21 Chromatin II
    Genetic Organization -Chromosomal Arrangement: From Form to Function. Chapters 9 & 10 in Genes XI The Eukaryotic chromosome – Organized Structures -banding – Centromeres – Telomeres – Nucleosomes – Euchromatin / Heterochromatin – Higher Orders of Chromosomal Structure 2 Heterochromatin differs from euchromatin in that heterochromatin is effectively inert; remains condensed during interphase; is transcriptionally repressed; replicates late in S phase and may be localized to the centromere or nuclear periphery Facultative heterochromatin is not restricted by pre-designated sequence; genes that are moved within or near heterochromatic regions can become inactivated as a result of their new location. Heterochromatin differs from euchromatin in that heterochromatin is effectively inert; remains condensed during interphase; is transcriptionally repressed; replicates late in S phase and may be localized to the centromere or nuclear periphery Facultative heterochromatin is not restricted by pre-designated sequence; genes that are moved within or near heterochromatic regions can become inactivated as a result of their new location. Chromatin inactivation (or heterochromatin formation) occurs by the addition of proteins to the nucleosomal fiber. May be due to: Chromatin condensation -making it inaccessible to transcriptional apparatus Proteins that accumulate and inhibit accessibility to the regulatory sequences Proteins that directly inhibit transcription Chromatin Is Fundamentally Divided into Euchromatin and Heterochromatin • Individual chromosomes can be seen only during mitosis. • During interphase, the general mass of chromatin is in the form of euchromatin, which is slightly less tightly packed than mitotic chromosomes. TF20210119 Regions of compact heterochromatin are clustered near the nucleolus and nuclear membrane Photo courtesy of Edmund Puvion, Centre National de la Recherche Scientifique Chromatin: Basic Structures • nucleosome – The basic structural subunit of chromatin, consisting of ~200 bp of DNA wrapped around an octamer of histone proteins.
    [Show full text]
  • Antibodies Products
    Chapter 2 : Gentaur Products List • Human Signal peptidase complex catalytic subunit • Human Sjoegren syndrome nuclear autoantigen 1 SSNA1 • Human Small proline rich protein 2A SPRR2A ELISA kit SEC11A SEC11A ELISA kit SpeciesHuman ELISA kit SpeciesHuman SpeciesHuman • Human Signal peptidase complex catalytic subunit • Human Sjoegren syndrome scleroderma autoantigen 1 • Human Small proline rich protein 2B SPRR2B ELISA kit SEC11C SEC11C ELISA kit SpeciesHuman SSSCA1 ELISA kit SpeciesHuman SpeciesHuman • Human Signal peptidase complex subunit 1 SPCS1 ELISA • Human Ski oncogene SKI ELISA kit SpeciesHuman • Human Small proline rich protein 2D SPRR2D ELISA kit kit SpeciesHuman • Human Ski like protein SKIL ELISA kit SpeciesHuman SpeciesHuman • Human Signal peptidase complex subunit 2 SPCS2 ELISA • Human Skin specific protein 32 C1orf68 ELISA kit • Human Small proline rich protein 2E SPRR2E ELISA kit kit SpeciesHuman SpeciesHuman SpeciesHuman • Human Signal peptidase complex subunit 3 SPCS3 ELISA • Human SLAIN motif containing protein 1 SLAIN1 ELISA kit • Human Small proline rich protein 2F SPRR2F ELISA kit kit SpeciesHuman SpeciesHuman SpeciesHuman • Human Signal peptide CUB and EGF like domain • Human SLAIN motif containing protein 2 SLAIN2 ELISA kit • Human Small proline rich protein 2G SPRR2G ELISA kit containing protein 2 SCUBE2 ELISA kit SpeciesHuman SpeciesHuman SpeciesHuman • Human Signal peptide CUB and EGF like domain • Human SLAM family member 5 CD84 ELISA kit • Human Small proline rich protein 3 SPRR3 ELISA kit containing protein
    [Show full text]
  • Single Cell Derived Clonal Analysis of Human Glioblastoma Links
    SUPPLEMENTARY INFORMATION: Single cell derived clonal analysis of human glioblastoma links functional and genomic heterogeneity ! Mona Meyer*, Jüri Reimand*, Xiaoyang Lan, Renee Head, Xueming Zhu, Michelle Kushida, Jane Bayani, Jessica C. Pressey, Anath Lionel, Ian D. Clarke, Michael Cusimano, Jeremy Squire, Stephen Scherer, Mark Bernstein, Melanie A. Woodin, Gary D. Bader**, and Peter B. Dirks**! ! * These authors contributed equally to this work.! ** Correspondence: [email protected] or [email protected]! ! Supplementary information - Meyer, Reimand et al. Supplementary methods" 4" Patient samples and fluorescence activated cell sorting (FACS)! 4! Differentiation! 4! Immunocytochemistry and EdU Imaging! 4! Proliferation! 5! Western blotting ! 5! Temozolomide treatment! 5! NCI drug library screen! 6! Orthotopic injections! 6! Immunohistochemistry on tumor sections! 6! Promoter methylation of MGMT! 6! Fluorescence in situ Hybridization (FISH)! 7! SNP6 microarray analysis and genome segmentation! 7! Calling copy number alterations! 8! Mapping altered genome segments to genes! 8! Recurrently altered genes with clonal variability! 9! Global analyses of copy number alterations! 9! Phylogenetic analysis of copy number alterations! 10! Microarray analysis! 10! Gene expression differences of TMZ resistant and sensitive clones of GBM-482! 10! Reverse transcription-PCR analyses! 11! Tumor subtype analysis of TMZ-sensitive and resistant clones! 11! Pathway analysis of gene expression in the TMZ-sensitive clone of GBM-482! 11! Supplementary figures and tables" 13" "2 Supplementary information - Meyer, Reimand et al. Table S1: Individual clones from all patient tumors are tumorigenic. ! 14! Fig. S1: clonal tumorigenicity.! 15! Fig. S2: clonal heterogeneity of EGFR and PTEN expression.! 20! Fig. S3: clonal heterogeneity of proliferation.! 21! Fig.
    [Show full text]
  • Supplementary Table 1
    Supplementary Table 1. 492 genes are unique to 0 h post-heat timepoint. The name, p-value, fold change, location and family of each gene are indicated. Genes were filtered for an absolute value log2 ration 1.5 and a significance value of p ≤ 0.05. Symbol p-value Log Gene Name Location Family Ratio ABCA13 1.87E-02 3.292 ATP-binding cassette, sub-family unknown transporter A (ABC1), member 13 ABCB1 1.93E-02 −1.819 ATP-binding cassette, sub-family Plasma transporter B (MDR/TAP), member 1 Membrane ABCC3 2.83E-02 2.016 ATP-binding cassette, sub-family Plasma transporter C (CFTR/MRP), member 3 Membrane ABHD6 7.79E-03 −2.717 abhydrolase domain containing 6 Cytoplasm enzyme ACAT1 4.10E-02 3.009 acetyl-CoA acetyltransferase 1 Cytoplasm enzyme ACBD4 2.66E-03 1.722 acyl-CoA binding domain unknown other containing 4 ACSL5 1.86E-02 −2.876 acyl-CoA synthetase long-chain Cytoplasm enzyme family member 5 ADAM23 3.33E-02 −3.008 ADAM metallopeptidase domain Plasma peptidase 23 Membrane ADAM29 5.58E-03 3.463 ADAM metallopeptidase domain Plasma peptidase 29 Membrane ADAMTS17 2.67E-04 3.051 ADAM metallopeptidase with Extracellular other thrombospondin type 1 motif, 17 Space ADCYAP1R1 1.20E-02 1.848 adenylate cyclase activating Plasma G-protein polypeptide 1 (pituitary) receptor Membrane coupled type I receptor ADH6 (includes 4.02E-02 −1.845 alcohol dehydrogenase 6 (class Cytoplasm enzyme EG:130) V) AHSA2 1.54E-04 −1.6 AHA1, activator of heat shock unknown other 90kDa protein ATPase homolog 2 (yeast) AK5 3.32E-02 1.658 adenylate kinase 5 Cytoplasm kinase AK7
    [Show full text]
  • A Temporally Controlled Sequence of X-Chromosome Inactivation and Reactivation Defines Female Mouse in Vitro Germ Cells with Meiotic Potential
    bioRxiv preprint doi: https://doi.org/10.1101/2021.08.11.455976; this version posted August 11, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A temporally controlled sequence of X-chromosome inactivation and reactivation defines female mouse in vitro germ cells with meiotic potential Jacqueline Severino1†, Moritz Bauer1,9†, Tom Mattimoe1, Niccolò Arecco1, Luca Cozzuto1, Patricia Lorden2, Norio Hamada3, Yoshiaki Nosaka4,5,6, So Nagaoka4,5,6, Holger Heyn2, Katsuhiko Hayashi7, Mitinori Saitou4,5,6 and Bernhard Payer1,8* Abstract The early mammalian germ cell lineage is characterized by extensive epigenetic reprogramming, which is required for the maturation into functional eggs and sperm. In particular, the epigenome needs to be reset before parental marks can be established and then transmitted to the next generation. In the female germ line, reactivation of the inactive X- chromosome is one of the most prominent epigenetic reprogramming events, and despite its scale involving an entire chromosome affecting hundreds of genes, very little is known about its kinetics and biological function. Here we investigate X-chromosome inactivation and reactivation dynamics by employing a tailor-made in vitro system to visualize the X-status during differentiation of primordial germ cell-like cells (PGCLCs) from female mouse embryonic stem cells (ESCs). We find that the degree of X-inactivation in PGCLCs is moderate when compared to somatic cells and characterized by a large number of genes escaping full inactivation.
    [Show full text]
  • Impairment of Spermatogenesis and Sperm Motility by the High-Fat Diet
    Gut microbiota ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2019-319127 on 2 January 2020. Downloaded from Impairment of spermatogenesis and sperm motility by the high- fat diet- induced dysbiosis of gut microbes Ning Ding ,1 Xin Zhang,2 Xue Di Zhang,1 Jun Jing,3,4 Shan Shan Liu,5 Yun Ping Mu,1 Li Li Peng ,6 Yun Jing Yan,1 Geng Miao Xiao,1 Xin Yun Bi,1 Hao Chen,1 Fang Hong Li,1 Bing Yao,3,4 Allan Z Zhao1 ► Additional material is ABSTRact published online only. To view, Objective High- fat diet (HFD)- induced metabolic Significance of this study please visit the journal online disorders can lead to impaired sperm production. We aim (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2019- 319127). to investigate if HFD- induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm ► High- fat diet (HFD) leads to obesity and 1The School of Biomedical motility. metabolic syndromes and affects the functions and Pharmaceutical Sciences, in reproductive system. Guangdong University of Design Faecal microbes derived from the HFD- fed or Technology, Guangzhou, normal diet (ND)- fed male mice were transplanted to the ► HFD- induced gut microbiota dysbiosis and Guangdong, China endotoxaemia have been reported. 2 mice maintained on ND. The gut microbes, sperm count Dizal Pharma, Shanghai, China Emerging evidence demonstrated that gut 3 and motility were analysed. Human faecal/semen/blood ► Jinling Hospital Department microbiota dysbiosis is closely associated Reproductive Medical Center, samples were collected to assess microbiota, sperm Nanjing Medicine University, quality and endotoxin.
    [Show full text]
  • The Characterization of Cell Surface Receptor Complexes by Affinity Chromatography, Liquid Chromatography and Tandem Mass Spectrometry
    The Characterization of Cell Surface Receptor Complexes by Affinity Chromatography, Liquid Chromatography and Tandem Mass Spectrometry by Jaimie Dufresne BSc, MSc, Ryerson University A dissertation presented to Ryerson University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program of Molecular Science Toronto, Ontario, Canada, 2017 © Jaimie Dufresne 2017 AUTHOR'S DECLARATION FOR ELECTRONIC SUBMISSION OF A DISSERTATION I hereby declare that I am the sole author of this dissertation. This is a true copy of the dissertation, including any required final revisions, as accepted by my examiners. I authorize Ryerson University to lend this dissertation to other institutions or individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this dissertation by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. I understand that my dissertation may be made electronically available to the public. ii Abstract Cell surface receptors are of critical importance to the treatment of disease but are difficult to isolate and identify by classical approaches. Here, a robust and general method for capturing a receptor complex from the surface of live cells with ligands presented on nanoscopic beads is demonstrated. Two forms of affinity chromatography: the presentation of a biotinylated ligand to the surface of live cells and recovered by classical affinity chromatography was compared to the presentation of the ligand on the surface of nanoscopic chromatography beads for the isolation of the IgG-FcR complex from the surface of live cells.
    [Show full text]
  • Content Based Search in Gene Expression Databases and a Meta-Analysis of Host Responses to Infection
    Content Based Search in Gene Expression Databases and a Meta-analysis of Host Responses to Infection A Thesis Submitted to the Faculty of Drexel University by Francis X. Bell in partial fulfillment of the requirements for the degree of Doctor of Philosophy November 2015 c Copyright 2015 Francis X. Bell. All Rights Reserved. ii Acknowledgments I would like to acknowledge and thank my advisor, Dr. Ahmet Sacan. Without his advice, support, and patience I would not have been able to accomplish all that I have. I would also like to thank my committee members and the Biomed Faculty that have guided me. I would like to give a special thanks for the members of the bioinformatics lab, in particular the members of the Sacan lab: Rehman Qureshi, Daisy Heng Yang, April Chunyu Zhao, and Yiqian Zhou. Thank you for creating a pleasant and friendly environment in the lab. I give the members of my family my sincerest gratitude for all that they have done for me. I cannot begin to repay my parents for their sacrifices. I am eternally grateful for everything they have done. The support of my sisters and their encouragement gave me the strength to persevere to the end. iii Table of Contents LIST OF TABLES.......................................................................... vii LIST OF FIGURES ........................................................................ xiv ABSTRACT ................................................................................ xvii 1. A BRIEF INTRODUCTION TO GENE EXPRESSION............................. 1 1.1 Central Dogma of Molecular Biology........................................... 1 1.1.1 Basic Transfers .......................................................... 1 1.1.2 Uncommon Transfers ................................................... 3 1.2 Gene Expression ................................................................. 4 1.2.1 Estimating Gene Expression ............................................ 4 1.2.2 DNA Microarrays ......................................................
    [Show full text]
  • University of Southampton Bioinformatic
    University of Southampton Faculty of Environmental and Life Sciences Biological Sciences Bioinformatic Analysis of Human Next Generation Sequencing Data; extracting Additional Information, Optimising Mapping and Variant Calling, and Application in a Rare Disease Volume 1 of 1 By Roshan Kumar Sood A thesis presented for the degree of Doctor of Philosophy May 2019 Bioinformatic Analysis of Human NGS Data; extracting Additional Info., Optimising Mapping & Variant Calling, Application in Rare Disease Roshan Kumar Sood Page 2 University of Southampton Abstract Faculty of Environmental and Life Science Biological Sciences A thesis presented for the degree of Doctor of Philosophy Bioinformatic Analysis of Human Next Generation Sequencing Data; extracting Additional Information, Optimising Mapping and Variant Calling, and Application in a Rare Disease by Roshan Kumar Sood With the increased application of Next Generation Sequencing (NGS) to medicine it is important to test and develop approaches to extract the optimum information from datasets. In this thesis five aspects of NGS are investigated ranging from quality control to variant calling. Firstly a method to estimate contamination from a VCF file was developed which would be useful in cases where no BAM file was available to use existing tools. Unmapped reads were investigated to extract additional information from NGS samples and were able to detect the abundance of oral microbes from saliva samples relative to blood collected samples, but failed to identify differences between inflammatory bowel disease patients and controls. For a familial trio with a reported rare case of Sedaghatian-type spondylometaphyseal dysplasia (SSMD) sequenced both by whole exome (WES) and genome (WGS) sequencing it was shown that nearly all coding variants from WES were called in WGS despite differences in mean depth of coverage.
    [Show full text]
  • Mitotic Checkpoints and Chromosome Instability Are Strong Predictors of Clinical Outcome in Gastrointestinal Stromal Tumors
    MITOTIC CHECKPOINTS AND CHROMOSOME INSTABILITY ARE STRONG PREDICTORS OF CLINICAL OUTCOME IN GASTROINTESTINAL STROMAL TUMORS. Pauline Lagarde1,2, Gaëlle Pérot1, Audrey Kauffmann3, Céline Brulard1, Valérie Dapremont2, Isabelle Hostein2, Agnès Neuville1,2, Agnieszka Wozniak4, Raf Sciot5, Patrick Schöffski4, Alain Aurias1,6, Jean-Michel Coindre1,2,7 Maria Debiec-Rychter8, Frédéric Chibon1,2. Supplemental data NM cases deletion frequency. frequency. deletion NM cases Mand between difference the highest setswith of theprobe a view isdetailed panel Bottom frequently. sorted totheless deleted theprobe are frequently from more and thefrequency deletion represent Yaxes inblue. are cases (NM) metastatic for non- frequencies Corresponding inmetastatic (red). probe (M)cases sets figureSupplementary 1: 100 100 20 40 60 80 20 40 60 80 0 0 chr14 1 chr14 88 chr14 175 chr14 262 chr9 -MTAP 349 chr9 -MTAP 436 523 chr9-CDKN2A 610 Histogram presenting the 2000 more frequently deleted deleted frequently the 2000 more presenting Histogram chr9-CDKN2A 697 chr9-CDKN2A 784 chr9-CDKN2B 871 chr9-CDKN2B 958 chr9-CDKN2B 1045 chr22 1132 chr22 1219 chr22 1306 chr22 1393 1480 1567 M NM 1654 1741 1828 1915 M NM GIST14 GIST2 GIST16 GIST3 GIST19 GIST63 GIST9 GIST38 GIST61 GIST39 GIST56 GIST37 GIST47 GIST58 GIST28 GIST5 GIST17 GIST57 GIST47 GIST58 GIST28 GIST5 GIST17 GIST57 CDKN2A Supplementary figure 2: Chromosome 9 genomic profiles of the 18 metastatic GISTs (upper panel). Deletions and gains are indicated in green and red, respectively; and color intensity is proportional to copy number changes. A detailed view is given (bottom panel) for the 6 cases presenting a homozygous 9p21 deletion targeting CDKN2A locus (dark green).
    [Show full text]
  • Dual Functions for Insulinoma-Associated 1 in Retinal Development
    University of Kentucky UKnowledge Theses and Dissertations--Biology Biology 2015 DUAL FUNCTIONS FOR INSULINOMA-ASSOCIATED 1 IN RETINAL DEVELOPMENT Marie A. Forbes-Osborne University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Forbes-Osborne, Marie A., "DUAL FUNCTIONS FOR INSULINOMA-ASSOCIATED 1 IN RETINAL DEVELOPMENT" (2015). Theses and Dissertations--Biology. 31. https://uknowledge.uky.edu/biology_etds/31 This Doctoral Dissertation is brought to you for free and open access by the Biology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Biology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies.
    [Show full text]