DOCSLIB.ORG

Microscopy-Based High-Content Analyses Identify Novel Chromosome Instability Genes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microscopy-Based High-Content Analyses Identify Novel Chromosome Instability Genes Microscopy-based High-content Analyses Identify Novel Chromosome Instability Genes including SKP1 by Laura L. Thompson A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba In partial fulfillment of the requirements of the degree of Doctor of Philosophy Department of Biochemistry and Medical Genetics University of Manitoba Winnipeg, Manitoba, Canada Copyright © 2018 by Laura Louise Thompson i ABSTRACT Cancer is a devastating disease responsible for ~80,000 deaths in Canada each year. Chromosome instability (CIN) is an abnormal phenotype frequently observed in cancer, characterized by an increase in the rate at which chromosomes or chromosomal fragments are gained or lost. CIN underlies the development of aggressive, drug resistant cancers, disease recurrence, and poor prognoses. Despite this, the majority of CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. This thesis describes the development and utilization of imaged-based assays designed to detect CIN phenotypes (i.e. nuclear size changes and micronucleus formation) following gene silencing. Mitotic chromosome spread analyses validated that changes in CIN phenotypes corresponded with numerical and structural chromosome defects by silencing the established CIN gene, SMC1A (Chapter 3). The assays were multiplexed in a high-content, siRNA-based screen of 164 candidate genes in hTERT and HT1080, which identified 148 putative CIN genes. Validation of 10 genes (e.g. SKP1) was performed in hTERT and HCT116, which confirmed gene silencing induced chromosome changes (Chapter 4). SKP1 is a component of the SKP1- CUL1-F-box protein (SCF) E3 ubiquitin ligase complex, which regulates degradation of numerous proteins within pathways that maintain chromosome stability. Accordingly, the aberrant biology underlying CIN in SKP1 silenced cells was investigated further using immunofluorescence, microscopy, and biochemical techniques (Chapter 5). A high-content screen of all 68 F-box genes was performed to determine which F-box proteins/SCF complexes regulate chromosome stability. EMI1, SKP2, and FBXL7 induced CIN phenotypes similar to SKP1, suggesting that the corresponding SCF complexes are required for chromosome stability. ii Increases in the levels of SCF substrates Survivin and CCNE1, were detected in SKP1 silenced cells. Replication stress, DNA damage, centromeric protein mislocalization, and centrosome defects were also observed, all of which are mechanisms known to underlie CIN. Significantly, a novel screen for CIN phenotypes was developed and employed in this study, which expedited human CIN gene identification and provided critical insights into the fundamental biological mechanisms that regulate chromosome stability. Importantly, characterizing the aberrant genes/mechanisms that underlie CIN and oncogenesis could ultimately reveal novel cancer therapeutic targets. iii ACKNOWLEDGEMENTS First, I would like to express my sincerest gratitude to my supervisor Dr. Kirk McManus for his time, mentorship, scientific advice, and continuous support throughout my graduate studies. I am extremely fortunate to have had the opportunity to work, learn, and grow in the McManus lab. I would also like to thank my advisory committee; Dr. Mark Nachtigal, Dr. Yvonne Myal, and Dr. Tamra Werbowetski-Ogilvie, for their critical assessments of my research project, challenging questions, valuable input, support, and career advice. I would like to acknowledge all members of the McManus lab, past and present; Zelda Lichtensztejn, Sajesh Babu, Brent Guppy, Amy Cisyk, Erin McAndrew, Yasamin Asbaghi, Lucile Jeusset, Nicole Wilson, Chloe Lepage, Manisha Bungsy, Claire Morden, and all summer and co-op students that I have had the pleasure of working with. Thank you for your friendship, support, and for making the McManus lab a wonderful place to work. I would especially like to thank Zelda, Sajesh and Brent for training me in the laboratory and providing extensive technical assistance and trouble-shooting advice. I am extremely grateful to Allison Baergen, my summer student of three years, for her hard work and significant contributions to this research project. This research would not have been possible without the financial support of NSERC, Research Manitoba, University of Manitoba, CancerCare Manitoba, Terry Fox Institute and the Research Institute in Oncology and Hematology. I would also like to thank the donors that provided funding for the Caroline A. Cope Award for Excellence in Oncology Research, Mindel Rady Olenick Fellowship in Human Genetics, Emil & Lynette Hain Oncology Award, Human Genetics Endowment Fund Award, Mindel & Tom Olenick Research Studentship in Medicine, Sheu L. Lee Family Scholarship in Oncology Research, Nancie J. Mauro Scholarship in Oncology Research, and Phyllis J. McAlpine Graduate Fellowship. I am truly grateful to my family; Dad, Mom, Steve, Debbie, Travis, Janene, Troy, Brianna, Rob, Lucille, Robbie, and Sarah for their encouragement and support in all aspects of my life and studies. Lastly, I would like to extend a special thank you to Mike for his unwavering love, patience, and support. Thank you for always being there for me. iv TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii ACKNOWLEDGEMENTS .................................................................................................... iv TABLE OF CONTENTS ..........................................................................................................v LIST OF ABBREVIATIONS ................................................................................................. ix LIST OF TABLES .................................................................................................................xiv LIST OF FIGURES ...............................................................................................................xvi USED WITH PERMISSION and CONTRIBUTION OF AUTHOR ............................... xviii RESEARCH RATIONALE AND HYPOTHESES..................................................................1 CHAPTER 1: INTRODUCTION .............................................................................................3 1.0.0. Cancer Burden ...................................................................................................................3 1.0.1. The Global and National Impact of Cancer ....................................................................3 1.0.2. Colorectal Cancer Burden ..............................................................................................4 1.1.0. Overview of Colorectal Cancer ..........................................................................................5 1.1.1. Clinical Features ............................................................................................................5 1.1.2. Risk Factors and Screening ............................................................................................6 1.1.3. Diagnosis and Staging ....................................................................................................7 1.1.4. Current Standard of Care ...............................................................................................9 1.2.0. Molecular Pathogenesis of Colorectal Cancer ....................................................................9 1.2.1. Genome Instability in Colorectal Cancer ...................................................................... 10 1.2.2. Chromosome Instability ............................................................................................... 12 1.3.0. Chromosome Instability in Yeast and Cross-Species Approaches .................................... 14 1.4.0. Biological Pathways that Maintain Chromosome Stability ............................................... 15 1.4.1. Sister Chromatid Cohesion........................................................................................... 16 1.4.2. Centromere and Kinetochore Protein Recruitment and Regulation ............................... 18 1.4.3. Centrosome Regulation and Microtubule Dynamics ..................................................... 21 1.4.4. Spindle Assembly Checkpoint ..................................................................................... 25 1.4.5. DNA Replication ......................................................................................................... 27 1.4.6. DNA Damage Response .............................................................................................. 30 1.4.7. Ubiquitin-dependent Proteasomal Degradation ............................................................ 32 1.5.0. The SCF Complex ........................................................................................................... 35 1.5.1. S-Phase Kinase Associated Protein 1 ........................................................................... 37 1.5.2. SKP1 Alterations in Cancer .......................................................................................... 39 1.6.0. CIN Phenotypes............................................................................................................... 40 1.6.1. Nuclear Size Changes as an Emerging Indicator for Large-scale DNA Content Changes in Cancer ..................................................................................................................... 40 1.6.2. Micronucleus
Load more

Recommended publications
  • Regulation and Dysregulation of Chromosome Structure in Cancer
    Regulation and Dysregulation of Chromosome Structure in Cancer The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Hnisz, Denes et al. “Regulation and Dysregulation of Chromosome Structure in Cancer.” Annual Review of Cancer Biology 2, 1 (March 2018): 21–40 © 2018 Annual Reviews As Published https://doi.org/10.1146/annurev-cancerbio-030617-050134 Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/117286 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ Regulation and dysregulation of chromosome structure in cancer Denes Hnisz1*, Jurian Schuijers1, Charles H. Li1,2, Richard A. Young1,2* 1 Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA * Corresponding authors Corresponding Authors: Denes Hnisz Whitehead Institute for Biomedical Research 455 Main Street Cambridge, MA 02142 Tel: (617) 258-7181 Fax: (617) 258-0376 [email protected] Richard A. Young Whitehead Institute for Biomedical Research 455 Main Street Cambridge, MA 02142 Tel: (617) 258-5218 Fax: (617) 258-0376 [email protected] 1 Summary Cancer arises from genetic alterations that produce dysregulated gene expression programs. Normal gene regulation occurs in the context of chromosome loop structures called insulated neighborhoods, and recent studies have shown that these structures are altered and can contribute to oncogene dysregulation in various cancer cells. We review here the types of genetic and epigenetic alterations that influence neighborhood structures and contribute to gene dysregulation in cancer, present models for insulated neighborhoods associated with the most prominent human oncogenes, and discuss how such models may lead to further advances in cancer diagnosis and therapy.
    [Show full text]
  • Gene Family: Gene Duplication and Retrotransposon Insertion
    21 Bucentaur (Bcnt)1 Gene Family: Gene Duplication and Retrotransposon Insertion Shintaro Iwashita and Naoki Osada Iwaki Meisei University / National Institute of Genetics Japan 1. Introduction Members of multiple gene families in higher organisms allow for more refined cellular signaling networks and structural organization toward more stable physiological homeostasis. Gene duplication is one the most powerful ways of providing an opportunity to create a novel gene(s) because a novel function might be acquired without the loss of the original gene function (Ohno, 1970). Gene duplication can result from unequal crossing over by recombination, retroposition of cDNA, or whole-genome duplication. Furthermore, a replication-based mechanism of change in gene copy number has been proposed recently (Hastings et al., 2009). Gene duplication generated by retroposition is frequently accompanied by deleterious effects because the insertion of cDNA into the genome is nearly random or unlinks the original gene location resulting in an alteration of the original vital functions of the target genes. Thus retroelements such as transposable elements and endogenous retroviruses have been thought of as “selfish”. On the other hand, gene duplication caused by unequal crossing over generally results in tandem alignment, which less frequently disrupts the functions of other genes. Recent genome-wide studies have demonstrated that retroelements can definitely contribute to the creation of individual novel genes and the modulation of gene expression, which allows for the dynamic diversity of biological systems, such as placental evolution (Rawn & Cross, 2008). It is now recognized that tandem duplication and retroposition are among the key factors that initiate the creation of novel gene family members (Brosius, 2005; Sorek, 2007; Kaessmann, 2010).
    [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]
  • Transcriptional Targets of Hepatocyte Growth Factor Signaling and Ki-Ras Oncogene Activation in Colorectal Cancer
    Oncogene (2006) 25, 91–102 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Transcriptional targets of hepatocyte growth factor signaling and Ki-ras oncogene activation in colorectal cancer IM Seiden-Long1,2, KR Brown1,2, W Shih1, DA Wigle3, N Radulovich1, I Jurisica1,2,4 and M-S Tsao1,2,5 1Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 3Department of Surgery, University of Toronto, Toronto, Ontario, Canada; 4Department of Computer Science, University of Toronto, Toronto, Ontario, Canada and 5Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Both Ki-ras mutation and hepatocyte growth factor Introduction (HGF) receptor Met overexpression occur at high frequency in colon cancer. This study investigates the Colorectal carcinogenesis is characterized by a well- transcriptional changes induced by Ki-ras oncogene and delineated series of genetic mutations and aberrant gene HGF/Met signaling activation in colon cancer cell lines in expression events (Fearon and Vogelstein, 1990). Ki-ras vitro and in vivo. The model system used in these studies oncogene activation and the overexpression of growth included the DLD-1 colon cancer cell line with a mutated factor receptors on the cell surface have been shown to Ki-ras allele, and the DKO-4 cell line generated from play important rolesin colon cancer progression DLD-1, with its mutant Ki-ras allele inactivated by (Shirasawa et al., 1993; Fazekas et al., 2000). Ki-ras is targeted disruption.
    [Show full text]
  • Dynamic Molecular Linkers of the Genome: the First Decade of SMC Proteins
    Downloaded from genesdev.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Dynamic molecular linkers of the genome: the first decade of SMC proteins Ana Losada1 and Tatsuya Hirano2,3 1Spanish National Cancer Center (CNIO), Madrid E-28029, Spain; 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA Structural maintenance of chromosomes (SMC) proteins in eukaryotes. The proposed actions of cohesin and con- are chromosomal ATPases, highly conserved from bac- densins offer a plausible, if not complete, explanation for teria to humans, that play fundamental roles in many the sudden appearance of thread-like “substances” (the aspects of higher-order chromosome organization and chromosomes) and their longitudinal splitting during dynamics. In eukaryotes, SMC1 and SMC3 act as the mitosis, first described by Walther Flemming (1882). core of the cohesin complexes that mediate sister chro- Remarkably, SMC proteins are conserved among the matid cohesion, whereas SMC2 and SMC4 function as three phyla of life, indicating that the basic strategy of the core of the condensin complexes that are essential chromosome organization may be evolutionarily con- for chromosome assembly and segregation. Another served from bacteria to humans. An emerging theme is complex containing SMC5 and SMC6 is implicated in that SMC proteins are dynamic molecular linkers of the DNA repair and checkpoint responses. The SMC com- genome that actively fold, tether, and manipulate DNA plexes form unique ring- or V-shaped structures with strands. Their diverse functions range far beyond chro- long coiled-coil arms, and function as ATP-modulated, mosome segregation, and involve nearly all aspects of dynamic molecular linkers of the genome.
    [Show full text]
  • Datasheet: VMA00937 Product Details
    Datasheet: VMA00937 Description: MOUSE ANTI CIAPIN1 Specificity: CIAPIN1 Format: Purified Product Type: PrecisionAb Monoclonal Clone: AB04/1G9 Isotype: IgG1 Quantity: 100 µl 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 Western Blotting 1/1000 The PrecisionAb label is reserved for antibodies that meet the defined performance criteria within Bio-Rad's ongoing antibody validation programme. Click here to learn how we validate our PrecisionAb range. Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Further optimization may be required dependent on sample type. Target Species Human Product Form Purified IgG - Liquid Preparation Mouse monoclonal antibody affinity purified on Protein G from tissue culture supernatant Buffer Solution Phosphate buffered saline Preservative 0.09% Sodium Azide Stabilisers Approx. Protein IgG concentration 1.0 mg/ml Concentrations Immunogen E. coli-derived recombinant protein of amino acids 1-312 of human CIAPIN1 Page 1 of 3 External Database Links UniProt: Q6FI81 Related reagents Entrez Gene: 57019 CIAPIN1 Related reagents Fusion Partners Spleen cells from immunised BALB/c mice were fused with cells of the mouse SP2/0 myeloma cell line Specificity Mouse anti CIAPIN1 antibody recognizes anamorsin, also known as cytokine-induced apoptosis inhibitor 1. CIAPIN1 is an electron transfer protein required for assembly of cytosolic iron-sulfur clusters, a family of cofactors critical for many cellular functions (Lipper et al.
    [Show full text]
  • Seq2pathway Vignette
    seq2pathway Vignette Bin Wang, Xinan Holly Yang, Arjun Kinstlick May 19, 2021 Contents 1 Abstract 1 2 Package Installation 2 3 runseq2pathway 2 4 Two main functions 3 4.1 seq2gene . .3 4.1.1 seq2gene flowchart . .3 4.1.2 runseq2gene inputs/parameters . .5 4.1.3 runseq2gene outputs . .8 4.2 gene2pathway . 10 4.2.1 gene2pathway flowchart . 11 4.2.2 gene2pathway test inputs/parameters . 11 4.2.3 gene2pathway test outputs . 12 5 Examples 13 5.1 ChIP-seq data analysis . 13 5.1.1 Map ChIP-seq enriched peaks to genes using runseq2gene .................... 13 5.1.2 Discover enriched GO terms using gene2pathway_test with gene scores . 15 5.1.3 Discover enriched GO terms using Fisher's Exact test without gene scores . 17 5.1.4 Add description for genes . 20 5.2 RNA-seq data analysis . 20 6 R environment session 23 1 Abstract Seq2pathway is a novel computational tool to analyze functional gene-sets (including signaling pathways) using variable next-generation sequencing data[1]. Integral to this tool are the \seq2gene" and \gene2pathway" components in series that infer a quantitative pathway-level profile for each sample. The seq2gene function assigns phenotype-associated significance of genomic regions to gene-level scores, where the significance could be p-values of SNPs or point mutations, protein-binding affinity, or transcriptional expression level. The seq2gene function has the feasibility to assign non-exon regions to a range of neighboring genes besides the nearest one, thus facilitating the study of functional non-coding elements[2]. Then the gene2pathway summarizes gene-level measurements to pathway-level scores, comparing the quantity of significance for gene members within a pathway with those outside a pathway.
    [Show full text]
  • Genetic and Genomic Analysis of Hyperlipidemia, Obesity and Diabetes Using (C57BL/6J × TALLYHO/Jngj) F2 Mice
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Nutrition Publications and Other Works Nutrition 12-19-2010 Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P. Stewart Marshall University Hyoung Y. Kim University of Tennessee - Knoxville, [email protected] Arnold M. Saxton University of Tennessee - Knoxville, [email protected] Jung H. Kim Marshall University Follow this and additional works at: https://trace.tennessee.edu/utk_nutrpubs Part of the Animal Sciences Commons, and the Nutrition Commons Recommended Citation BMC Genomics 2010, 11:713 doi:10.1186/1471-2164-11-713 This Article is brought to you for free and open access by the Nutrition at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Nutrition Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Stewart et al. BMC Genomics 2010, 11:713 http://www.biomedcentral.com/1471-2164/11/713 RESEARCH ARTICLE Open Access Genetic and genomic analysis of hyperlipidemia, obesity and diabetes using (C57BL/6J × TALLYHO/JngJ) F2 mice Taryn P Stewart1, Hyoung Yon Kim2, Arnold M Saxton3, Jung Han Kim1* Abstract Background: Type 2 diabetes (T2D) is the most common form of diabetes in humans and is closely associated with dyslipidemia and obesity that magnifies the mortality and morbidity related to T2D. The genetic contribution to human T2D and related metabolic disorders is evident, and mostly follows polygenic inheritance. The TALLYHO/ JngJ (TH) mice are a polygenic model for T2D characterized by obesity, hyperinsulinemia, impaired glucose uptake and tolerance, hyperlipidemia, and hyperglycemia.
    [Show full text]
  • 2020 Program Book
    PROGRAM BOOK Note that TAGC was cancelled and held online with a different schedule and program. This document serves as a record of the original program designed for the in-person meeting. April 22–26, 2020 Gaylord National Resort & Convention Center Metro Washington, DC TABLE OF CONTENTS About the GSA ........................................................................................................................................................ 3 Conference Organizers ...........................................................................................................................................4 General Information ...............................................................................................................................................7 Mobile App ....................................................................................................................................................7 Registration, Badges, and Pre-ordered T-shirts .............................................................................................7 Oral Presenters: Speaker Ready Room - Camellia 4.......................................................................................7 Poster Sessions and Exhibits - Prince George’s Exhibition Hall ......................................................................7 GSA Central - Booth 520 ................................................................................................................................8 Internet Access ..............................................................................................................................................8
    [Show full text]
  • Down Regulation of CIAPIN1 Reverses Multidrug Resistance in Human Breast Cancer Cells by Inhibiting MDR1
    Molecules 2012, 17, 7595-7611; doi:10.3390/molecules17067595 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Down Regulation of CIAPIN1 Reverses Multidrug Resistance in Human Breast Cancer Cells by Inhibiting MDR1 Dan Lu 1,†,*, Zhibo Xiao 2,†, Wenxiu Wang 1, Yuqing Xu 1, Shujian Gao 1, Lili Deng 1, Wen He 1, Yu Yang 1, Xiaofei Guo 1 and Xuemei Wang 1 1 Department of Oncology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China 2 Department of Plastic Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]. Received: 13 March 2012; in revised form: 11 June 2012 / Accepted: 11 June 2012 / Published: 20 June 2012 Abstract: Cytokine-induced apoptosis inhibitor 1 (CIAPIN1), initially named anamorsin, a newly indentified antiapoptotic molecule is a downstream effector of the receptor tyrosine kinase-Ras signaling pathway. Current study has revealed that CIAPIN1 may have wide and important functions, especially due to its close correlations with malignant tumors. However whether or not it is involved in the multi-drug resistance (MDR) process of breast cancer has not been elucidated. To explore the effect of CIAPIN1 on MDR, we examined the expression of P-gp and CIAPIN1 by immunohistochemistry and found there was positive correlation between them. Then we successfully interfered with RNA translation by the infection of siRNA of CIAPIN1 into MCF7/ADM breast cancer cell lines through a lentivirus, and the expression of the target gene was significantly inhibited.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Noelia Díaz Blanco
    Effects of environmental factors on the gonadal transcriptome of European sea bass (Dicentrarchus labrax), juvenile growth and sex ratios Noelia Díaz Blanco Ph.D. thesis 2014 Submitted in partial fulfillment of the requirements for the Ph.D. degree from the Universitat Pompeu Fabra (UPF). This work has been carried out at the Group of Biology of Reproduction (GBR), at the Department of Renewable Marine Resources of the Institute of Marine Sciences (ICM-CSIC). Thesis supervisor: Dr. Francesc Piferrer Professor d’Investigació Institut de Ciències del Mar (ICM-CSIC) i ii A mis padres A Xavi iii iv Acknowledgements This thesis has been made possible by the support of many people who in one way or another, many times unknowingly, gave me the strength to overcome this "long and winding road". First of all, I would like to thank my supervisor, Dr. Francesc Piferrer, for his patience, guidance and wise advice throughout all this Ph.D. experience. But above all, for the trust he placed on me almost seven years ago when he offered me the opportunity to be part of his team. Thanks also for teaching me how to question always everything, for sharing with me your enthusiasm for science and for giving me the opportunity of learning from you by participating in many projects, collaborations and scientific meetings. I am also thankful to my colleagues (former and present Group of Biology of Reproduction members) for your support and encouragement throughout this journey. To the “exGBRs”, thanks for helping me with my first steps into this world. Working as an undergrad with you Dr.
    [Show full text]