DOCSLIB.ORG

Characterization of Gonadal Transcriptomes from the Turbot (Scophthalmus Maximus)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of Gonadal Transcriptomes from the Turbot (Scophthalmus Maximus) Genome Characterization of Gonadal Transcriptomes from the Turbot (Scophthalmus maximus) Journal: Genome Manuscript ID gen-2014-0190.R3 Manuscript Type: Article Date Submitted by the Author: 02-Oct-2015 Complete List of Authors: Hu, Yulong; Yellow Sea Fisheries Research Institute (YSFRI), Huang, Meng; cornell university, Wang, Weiji; Yellow Sea Fisheries Research Institute (YSFRI), Guan, Jiantao;Draft Yellow Sea Fisheries Research Institute (YSFRI), Kong, Jie; Yellow Sea Fisheries Research Institute (YSFRI), Keyword: Turbot, Transcriptome, sex, SNP Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online. gen-2014-0190.R3 Supplement1-Dataset S1.rar gen-2014-0190.R3 Supplement2-Dataset S2.zip https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 701 Genome Characterization of Gonadal Transcriptomes from the Turbot ( Scophthalmus maximus ) Yulong Hu #, Meng Huang #, Weiji Wang, Jiantao Guan, Jie Kong* Yellow Sea Fisheries Research Institute (YSFRI), Chinese Academy of Fishery Sciences, Qingdao, China Draft Corresponding author: Jie Kong, PhD Yellow Sea Fisheries Research Institute (YSFRI) Chinese Academy of Fishery Sciences Qingdao, China Phone: 0086-532-85821650 Fax: 0086 053285811514 Email: [email protected] 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 701 Abstract The mechanisms underlying sexual reproduction and sex ratio determination remains unclear in turbot, a flatfish of great commercial value. And there is limited information in the turbot database regarding genes related to the reproductive system. Here, we conducted high-throughput transcriptome profiling of turbot gonad tissues to better understand their reproductive functions and to supply essential gene sequence information for marker-assisted selection programs in the turbot industry. In this study, two gonad libraries representing sex differences in S.maximus yielded 453,818 high-quality reads that were assembled into 24,611 contigs and 33,713 singletons by using 454 pyrosequencing, 13,936Draft CS (contigs and singletons) of which were annotated using BLASTx. GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses, revealed that various biological functions and processes were associated with many of the annotated CS (contigs and singletons). Expression analyses showed that 510 genes were differentially expressed in males versus females; 80% of these genes were annotated. In addition, 6,484 and 6,036 single nucleotide polymorphisms (SNPs) were identified in males and females libraries, respectively. This transcriptome resource will serve as the foundation for cDNA or SNP microarray construction, gene expression characterization, and sex-specific linkage mapping in turbot. Key words: Turbot; Transcriptome; sex; SNP 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 701 Genome Introduction The turbot ( Scophthalmus maximus ), also known as Psetta maxima, is an important economic flatfish species belonging to the family Scophthalmidae (order Pleuronectiformes). It is a marine flatfish species naturally distributed all along the European coasts, from Norway to Morocco in the Atlantic area, and across the northern coasts of the Mediterranean up to the Black Sea (Blanquer et al. 1992). Following, it was introduced to China as well for farming purposes in 1992 (Lei 2003). In 2012, turbot aquaculture production was 12,676 tons in Europe, while 64,000 tons in China, which represents a world leader in adult turbot production (FAO 2014). During intensive farming, turbot females can exhibit over two-fold increase in their growth rate as compared to malesDraft (Husebye et al. 1994). This is among the highest sex-related differential growth rates in cultured marine fish (Piferrer et al. 1995). The difference in the growth rate could be observed as early as 8 months after hatching (Imsland et al. 1997), and it is maintained throughout the production cycle, even following sexual maturation (Piferrer et al. 2004). Under culture conditions, the males also mature earlier than females, which can reduce somatic growth and increase susceptibility to diseases and mortality (Imsland et al. 1997; Piferrer et al. 2003). Altogether, these factors result in loss of production during the grow-out phase and, therefore, all-female populations should be established to optimize turbot production. The sex ratio depends on processes related to both genetic and environmental factors, which are directly related to the reproductive potential of the population (Penman et al. 2008). Flatfish have been reported to possess genetic systems ranging from polygenic to dominant sex-determining factors that are influenced by autosomal controls in male (XX/XY) or female heterogametic systems (ZZ/ZW) (Purdom 1972; Lincoln 1981; Tabata 1991; Howell et al. 1995; Aida and Arai 1998; Goto et al. 1999; 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 701 Tvedt et al. 2006 ). The most common mode of GSD is determined by the inherited combination of sex-determining genes or polygenic systems (Oldfield 2005 ). Recently, the number of identified expressed sequence tags (ESTs) has increased very rapidly in turbot, it remains insufficient when compared with some other teleost (e.g. 15,598 vs 301,380; Salmon, GenBank/NCBI). Although several sex-associated markers have been discovered (Casas et al. 2005 ) and some linkage maps constructed or enriched (Bouza et al. 2007; Bouza et al. 2008; Bouza et al. 2012; Martínez et al. 2008 ), the sex-determining gene remains not defined and precisely sex-associated linkage maps are not available. However, development of a database of genes sequences has led to the identification of the major sex-determining region (Martínez et al. 2009 ) and mapping of DNA sex-specific markers and genes related to sex differentiation in turbot (Viñas et al. 2012). Draft RNA sequencing (RNA-seq) was used for transcriptome profiling, allowing us to rapidly identify the vast majority of transcriptomes and new splicing variants in a cost-effective manner. The Genome Sequencer FLX (GS FLX) System, powered by 454 sequencing technology, was selected as the sequencing platform because this technology is more appropriate than other next-generation sequencing platforms (such as the Illumina Solexa and ABI SOLiD) for de novo transcriptome sequencing in non-model organisms (Vera et al. 2008; Meyer et al. 2009; O’Neil et al. 2010 ). In particular, many transcriptome profiles of some marine species have been successfully mapped by using 454 sequencing (Kawahara-Miki et al. 2011; Milan et al. 2011; Hou et al. 2011; Du et al. 2012; Pereiro et al. 2012 ). Recent studies have reported large-scale transcriptome profiling of S.maximus using 454 sequencing to identify candidate genes involved in immunity and sex differentiation (Pereiro et al. 2012; Rabis et al. 2013 ). Although the data (more than 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 701 Genome 120,876 contigs and 237,580 singletons) obtained from those tissues (brain, hypophysis, gonad, gill, liver, spleen and head kidney) had found some genes involved in sex differentiation and maturation, more sequencing information is still needed to identify novel, key genes involved in gonad-specific or sex-associated genes. To supplement the genomic resources of the turbot, especially with respect to the sex transcriptome, we used a 454 pyrosequencing method to identify differentially expressed genes and potential genetic markers in gonad tissues. The data generated in this study will facilitate further research examining sex- and reproductive-related genes in the turbot, which will be useful for linkage map construction. Materials and methods Draft Ethics Statement The Institutional Animal Care and Use Committee (IACUC) of the Yellow Sea Fisheries Research Institute (YSFRI) approved this animal study. All of the fishes were sacrificed by an overdose of anesthesia to minimize suffering, and the experimental procedures were performed according to the institutional guidelines of the State Council of the People’s Republic of China. Turbot sampling Gonad tissues from 12 male and 12 female turbot (aged 600 days) were collected for 454 sequencing. Artificial fertilization and maintenance of the larval cultures were performed by the Huanghai Aquatic Product Group Co., Ltd. (Haiyang, Shandong Province, China). Twenty juvenile fishes (aged 150 days) were provided by the Laboratory of Genetic Resources and Breeding for independent SNP validation at the Yellow Sea Fisheries Research Institute (Qingdao, Shandong Province, China). The analyzed turbot represented a mixture of unrelated families, and the number, age and 5 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 701 mean value of morphometric data (standard length, width and weight) for each animal group were as follows: adult males(n=12; 600 days post-hatch; 29.5±1.6 cm, 24.0±1.0 cm, 1398.7±88.4 g); adult females ( n=12; 600 days post-hatch; 30.7±0.9 cm, 25.0±0.6 cm, 1519.8±165.7 g); juvenile males ( n=10; 150 days post-hatch; 8.8±1.1 cm, 6.7±1.0 cm, 28.2±11.0 g); juvenile females ( n=10; 150 days post-hatch; 9.3±0.6 cm, 7.3±0.4 cm, 36.2±7.8 g). Gonads were removed from all turbot individuals for 454 sequencing and sex identification, and pterygiophore tissues were removed from juvenile turbot for SNP validation. For sex identification, the tissues were stained with hematoxylinand eosin, embedded in paraffin and sliced into 6-µm sections. Phenotypic sex identification was conducted by microscopic observation. All of the samples subjected to 454 sequencing and SNP validation were flash frozen in liquid nitrogen and stored at -80 °C untilDraft RNA extraction. Total RNA extraction, cDNA library construction and 454 GS-FLX sequencing Total RNA was extracted and purified from each tissue using the RNA prep pure Tissue Kit (Tiangen Biotech (Beijing) Co., Ltd., China) according to the manufacturer’s protocol. The quantity and quality of the total RNA was assessed using an Ultrospec TM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by gel electrophoresis.
Load more

Recommended publications
  • Small Cell Ovarian Carcinoma: Genomic Stability and Responsiveness to Therapeutics
    Gamwell et al. Orphanet Journal of Rare Diseases 2013, 8:33 http://www.ojrd.com/content/8/1/33 RESEARCH Open Access Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics Lisa F Gamwell1,2, Karen Gambaro3, Maria Merziotis2, Colleen Crane2, Suzanna L Arcand4, Valerie Bourada1,2, Christopher Davis2, Jeremy A Squire6, David G Huntsman7,8, Patricia N Tonin3,4,5 and Barbara C Vanderhyden1,2* Abstract Background: The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer. Method: The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested. Results: BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing.
    [Show full text]
  • Chromosomal Aberrations in Head and Neck Squamous Cell Carcinomas in Norwegian and Sudanese Populations by Array Comparative Genomic Hybridization
    825-843 12/9/08 15:31 Page 825 ONCOLOGY REPORTS 20: 825-843, 2008 825 Chromosomal aberrations in head and neck squamous cell carcinomas in Norwegian and Sudanese populations by array comparative genomic hybridization ERIC ROMAN1,2, LEONARDO A. MEZA-ZEPEDA3, STINE H. KRESSE3, OLA MYKLEBOST3,4, ENDRE N. VASSTRAND2 and SALAH O. IBRAHIM1,2 1Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen, Jonas Lies vei 91; 2Department of Oral Sciences - Periodontology, Faculty of Medicine and Dentistry, University of Bergen, Årstadveien 17, 5009 Bergen; 3Department of Tumor Biology, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, Montebello, 0310 Oslo; 4Department of Molecular Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway Received January 30, 2008; Accepted April 29, 2008 DOI: 10.3892/or_00000080 Abstract. We used microarray-based comparative genomic logical parameters showed little correlation, suggesting an hybridization to explore genome-wide profiles of chromosomal occurrence of gains/losses regardless of ethnic differences and aberrations in 26 samples of head and neck cancers compared clinicopathological status between the patients from the two to their pair-wise normal controls. The samples were obtained countries. Our findings indicate the existence of common from Sudanese (n=11) and Norwegian (n=15) patients. The gene-specific amplifications/deletions in these tumors, findings were correlated with clinicopathological variables. regardless of the source of the samples or attributed We identified the amplification of 41 common chromosomal carcinogenic risk factors. regions (harboring 149 candidate genes) and the deletion of 22 (28 candidate genes). Predominant chromosomal alterations Introduction that were observed included high-level amplification at 1q21 (harboring the S100A gene family) and 11q22 (including Head and neck squamous cell carcinoma (HNSCC), including several MMP family members).
    [Show full text]
  • Interacción Célula Sinovial-Fibrina Como Mecanismo Patogénico Y Oportunidad Terapéutica En Artritis Reumatoide
    UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS DEPARTAMENTO DE BIOQUÍMICA Y BIOLOGÍA MOLECULAR I TESIS DOCTORAL Interacción célula sinovial-fibrina como mecanismo patogénico y oportunidad terapéutica en artritis reumatoide MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Antonio Gabucio López DIRECTORA Olga Sánchez Pernaute Madrid, 2017 © Antonio Gabucio López, 2016 Universidad Complutense de Madrid FACULTAD DE CIENCIAS QUÍMICAS TESIS DOCTORAL Interacción célula sinovial-fibrina como mecanismo patogénico y oportunidad terapéutica en artritis reumatoide PRESENTADA POR Antonio Gabucio López Facultad de Ciencias Químicas Directora Olga Sánchez Pernaute I A todos los que me han ayudado por hacer la vida más fácil y a todos los que me han “des”-ayudado por enseñarme a ser más fuerte II III AGRADECIMIENTOS A la Dra. Olga Sánchez Pernaute, directora del estudio, por su incansable apoyo y asesoramiento científico. Al doctor Gabriel Herrero-Beaumont Cuenca, jefe de servicio de reumatología de la Fundación Jiménez Díaz, por su insustituible apoyo y sin el cual, esta tesis no hubiera sido posible. A los pacientes anónimos, objeto y objetivo del estudio, sin cuya existencia y solidaridad nada de esto tendría sentido. A la Sociedad Española de Reumatología, por el apoyo prestado en el origen de este proyecto. A la Fundación Conchita Rábago de Jiménez Díaz por su soporte financiero. A todas aquellas personas que de una u otra manera han contribuido al desarrollo de esta tesis doctoral. IV V ÍNDICE RESUMEN / ABSTRACT..............................................................................................
    [Show full text]
  • Aplicación De La Biología De Sistemas Al Estudio De La Malaria Y Búsqueda De Biomarcadores Y Dianas Terapéuticas
    Aplicación de la biología de sistemas al estudio de la malaria y búsqueda de biomarcadores y dianas terapéuticas Autora: Mireia Ferrer Almirall Máster en Bioinformática y Bioestadística Area 1-Bioinformática farmacéutica Tutores: Melchor Sánchez Martínez y Alex Sánchez Pla Profesor responsable de la asignatura: Carles Ventura Royo 02/01/2019 Esta obra está sujeta a una licencia de Reconocimiento-NoComercial- SinObraDerivada 3.0 España de Creative Commons FICHA DEL TRABAJO FINAL Aplicación de la biología de sistemas al Título del trabajo: estudio de la malaria y búsqueda de biomarcadores y dianas terapéuticas Nombre del autor: Mireia Ferrer Almirall Melchor Sánchez Martínez y Nombre del consultor/a: Alex Sánchez Pla Nombre del PRA: Carles Ventura Royo Fecha de entrega (mm/aaaa): 01/2019 Titulación: Máster en Bioinformática y Bioestadística Área del Trabajo Final: 1-Bioinformática farmacéutica Idioma del trabajo: castellano Malaria, Biología-de-sistemas, Palabras clave dianas-terapéuticas Resumen del Trabajo (máximo 250 palabras): Con la finalidad, contexto de aplicación, metodología, resultados i conclusiones del trabajo. La finalidad de este trabajo es aplicar herramientas de biología de sistemas para investigar los mecanismos implicados en la infección por el parásito de la malaria e identificar posibles biomarcadores y dianas terapéuticas. Se ha partido de una serie temporal de datos de microarrays del bazo de ratones infectados con dos cepas del parásito (NL y L) para determinar los genes que se encuentran diferencialmente expresados (DEG) respecto a ratones control. A partir de las listas de DEG obtenidas, se han utilizado herramientas de biología de sistemas en combinación con análisis de significación biológica para obtener una visión integrada de los procesos biológicos que se encuentran alterados en la enfermedad e identificar posibles biomarcadores/dianas terapéuticas.
    [Show full text]
  • Stranded DNA and Sensitizes Human Kidney Renal Clear Cell Carcinoma
    RESEARCH ARTICLE Exosome component 1 cleaves single- stranded DNA and sensitizes human kidney renal clear cell carcinoma cells to poly(ADP-ribose) polymerase inhibitor Qiaoling Liu1†, Qi Xiao1†, Zhen Sun1†, Bo Wang2†, Lina Wang1, Na Wang1, Kai Wang1, Chengli Song1*, Qingkai Yang1* 1Institute of Cancer Stem Cell, DaLian Medical University, Dalian, China; 2Department of General Surgery, Second Affiliated Hospital, DaLian Medical University, Dalian, China Abstract Targeting DNA repair pathway offers an important therapeutic strategy for Homo sapiens (human) cancers. However, the failure of DNA repair inhibitors to markedly benefit patients necessitates the development of new strategies. Here, we show that exosome component 1 (EXOSC1) promotes DNA damages and sensitizes human kidney renal clear cell carcinoma (KIRC) cells to DNA repair inhibitor. Considering that endogenous source of mutation (ESM) constantly assaults genomic DNA and likely sensitizes human cancer cells to the inhibitor, we first analyzed the statistical relationship between the expression of individual genes and the mutations for KIRC. Among the candidates, EXOSC1 most notably promoted DNA damages and subsequent mutations via preferentially cleaving C site(s) in single-stranded DNA. Consistently, EXOSC1 was more *For correspondence: significantly correlated with C>A transversions in coding strands than these in template strands in [email protected] human KIRC. Notably, KIRC patients with high EXOSC1 showed a poor prognosis, and EXOSC1 (CS); sensitized human cancer cells to poly(ADP-ribose) polymerase inhibitors. These results show that [email protected] (QY) EXOSC1 acts as an ESM in KIRC, and targeting EXOSC1 might be a potential therapeutic strategy. †These authors contributed equally to this work Competing interests: The Introduction authors declare that no DNA damages and subsequent mutations are central to development, progression, and treatment competing interests exist.
    [Show full text]
  • ITRAQ-Based Quantitative Proteomic Analysis of Processed Euphorbia Lathyris L
    Zhang et al. Proteome Science (2018) 16:8 https://doi.org/10.1186/s12953-018-0136-6 RESEARCH Open Access ITRAQ-based quantitative proteomic analysis of processed Euphorbia lathyris L. for reducing the intestinal toxicity Yu Zhang1, Yingzi Wang1*, Shaojing Li2*, Xiuting Zhang1, Wenhua Li1, Shengxiu Luo1, Zhenyang Sun1 and Ruijie Nie1 Abstract Background: Euphorbia lathyris L., a Traditional Chinese medicine (TCM), is commonly used for the treatment of hydropsy, ascites, constipation, amenorrhea, and scabies. Semen Euphorbiae Pulveratum, which is another type of Euphorbia lathyris that is commonly used in TCM practice and is obtained by removing the oil from the seed that is called paozhi, has been known to ease diarrhea. Whereas, the mechanisms of reducing intestinal toxicity have not been clearly investigated yet. Methods: In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method was applied to investigate the effects of Euphorbia lathyris L. on the protein expression involved in intestinal metabolism, in order to illustrate the potential attenuated mechanism of Euphorbia lathyris L. processing. Differentially expressed proteins (DEPs) in the intestine after treated with Semen Euphorbiae (SE), Semen Euphorbiae Pulveratum (SEP) and Euphorbiae Factor 1 (EFL1) were identified. The bioinformatics analysis including GO analysis, pathway analysis, and network analysis were done to analyze the key metabolic pathways underlying the attenuation mechanism through protein network in diarrhea. Western blot were performed to validate selected protein and the related pathways. Results: A number of differentially expressed proteins that may be associated with intestinal inflammation were identified.
    [Show full text]
  • A Yeast Phenomic Model for the Influence of Warburg Metabolism on Genetic Buffering of Doxorubicin Sean M
    Santos and Hartman Cancer & Metabolism (2019) 7:9 https://doi.org/10.1186/s40170-019-0201-3 RESEARCH Open Access A yeast phenomic model for the influence of Warburg metabolism on genetic buffering of doxorubicin Sean M. Santos and John L. Hartman IV* Abstract Background: The influence of the Warburg phenomenon on chemotherapy response is unknown. Saccharomyces cerevisiae mimics the Warburg effect, repressing respiration in the presence of adequate glucose. Yeast phenomic experiments were conducted to assess potential influences of Warburg metabolism on gene-drug interaction underlying the cellular response to doxorubicin. Homologous genes from yeast phenomic and cancer pharmacogenomics data were analyzed to infer evolutionary conservation of gene-drug interaction and predict therapeutic relevance. Methods: Cell proliferation phenotypes (CPPs) of the yeast gene knockout/knockdown library were measured by quantitative high-throughput cell array phenotyping (Q-HTCP), treating with escalating doxorubicin concentrations under conditions of respiratory or glycolytic metabolism. Doxorubicin-gene interaction was quantified by departure of CPPs observed for the doxorubicin-treated mutant strain from that expected based on an interaction model. Recursive expectation-maximization clustering (REMc) and Gene Ontology (GO)-based analyses of interactions identified functional biological modules that differentially buffer or promote doxorubicin cytotoxicity with respect to Warburg metabolism. Yeast phenomic and cancer pharmacogenomics data were integrated to predict differential gene expression causally influencing doxorubicin anti-tumor efficacy. Results: Yeast compromised for genes functioning in chromatin organization, and several other cellular processes are more resistant to doxorubicin under glycolytic conditions. Thus, the Warburg transition appears to alleviate requirements for cellular functions that buffer doxorubicin cytotoxicity in a respiratory context.
    [Show full text]
  • A Chemosensitization Screen Identifies TP53RK, a Kinase That Restrains Apoptosis After Mitotic Stress
    Published OnlineFirst July 20, 2010; DOI: 10.1158/0008-5472.CAN-10-0015 Published OnlineFirst on July 20, 2010 as 10.1158/0008-5472.CAN-10-0015 Tumor and Stem Cell Biology Cancer Research A Chemosensitization Screen Identifies TP53RK, a Kinase that Restrains Apoptosis after Mitotic Stress David Peterson1, James Lee2, Xingye C. Lei3, William F. Forrest3, David P. Davis2, Peter K. Jackson1, and Lisa D. Belmont1 Abstract Taxanes are very effective at causing mitotic arrest; however, there is variability among cancer cells in the apoptotic response to mitotic arrest. The variability in clinical efficacy of taxane-based therapy is likely a re- flection of this variability in apoptotic response, thus elucidation of the molecular mechanism of the apoptotic response to mitotic stress could lead to improved clinical strategies. To identify genes whose expression in- fluences the rate and extent of apoptosis after mitotic arrest, we screened a kinase-enriched small interfering RNA library for effects on caspase activation in response to maximally effective doses of paclitaxel, a PLK1 inhibitor, or cisplatin. Small interfering RNA oligonucleotides directed against an atypical protein kinase, TP53RK, caused the greatest increase in caspase-3/7 activation in response to antimitotic agents. Time-lapse microscopy revealed that cells entered mitosis with normal kinetics, but died after entry into mitosis in the presence of paclitaxel more rapidly when TP53RK was depleted. Because expression levels of TP53RK vary in cancers, TP53RK levels could provide a molecular marker to predict response to antimitotic agents. TP53RK inhibition may also sensitize cancers to taxanes. Cancer Res; 70(15); 6325–35.
    [Show full text]
  • Rna-Sequencing Applications: Gene Expression Quantification and Methylator Phenotype Identification
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 8-2013 RNA-SEQUENCING APPLICATIONS: GENE EXPRESSION QUANTIFICATION AND METHYLATOR PHENOTYPE IDENTIFICATION Guoshuai Cai Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Bioinformatics Commons, Computational Biology Commons, and the Medicine and Health Sciences Commons Recommended Citation Cai, Guoshuai, "RNA-SEQUENCING APPLICATIONS: GENE EXPRESSION QUANTIFICATION AND METHYLATOR PHENOTYPE IDENTIFICATION" (2013). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 386. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/386 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. RNA-SEQUENCING APPLICATIONS: GENE EXPRESSION QUANTIFICATION AND METHYLATOR PHENOTYPE IDENTIFICATION
    [Show full text]
  • Namyoung Jung
    EPIGENETIC BASIS OF STEM CELL IDENTITY IN NORMAL AND MALIGNANT HEMATOPOIETIC DEVELOPMENT by Namyoung Jung A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July, 2015 © 2015 Namyoung Jung All Rights Reserved Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by subpopulations of leukemia-initiating or leukemia stem cells (LSC) that give rise to clonally related non-stem leukemic blasts. The LSC model proposes that since LSC and their blast progeny are clonally related, their functional properties must be due to epigenetic differences. In addition, the cell of origin of LSC among normal hematopoietic stem and progenitor cells (HSPCs) has yet to be clearly demonstrated. In order to investigate the role of epigenetics in LSC function and hematopoietic development, we profiled DNA methylation and gene expression of CD34+CD38-, CD34+CD38+ and CD34- cells from 15 AML patients, along with 6 well-defined HSPC populations from 5 normal bone marrows using Illumina Infinium HumanMethylation450 BeadChip and Affymetrix Human Genome U133 Plus 2.0 Array. To define LSC and blast functionally, we performed engraftment assays on the three subpopulations from 15 AML patients and defined 20 LSCs and 24 blast samples. We identified the key functional LSC epigenetic signature able to distinguish LSC from blasts that consisted of 84 differential methylations regions (DMRs) in 70 genes that correlated with differential gene expression. HOXA cluster genes were enriched within the LSC epigenetic signature. We found that most of these DMRs involve epigenetic alteration independent of underlying mutations, although several are downstream targets of genetic mutation in epigenome modifying enzymes and upstream regulators.
    [Show full text]
  • Screen for Kinases Affecting Amyloidogenic Cleavage by BACE1
    Screen for kinases affecting amyloidogenic cleavage by BACE1 Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie vorgelegt von Stephan Penzkofer Konstanz, Juli 2011 Tag der mündlichen Prüfung: 24.10.2011 1. Referent: Professor Dr. Marcel Leist 2. Referent: Professor Dr. Daniel Dietrich Summary: The Amyloid β peptide (Aβ) is suspected to be a causal agent for Alzheimer’s disease (AD). Therefore a screen for kinases downregulating the initial step of its production, the cleavage of the Amyloid Precursor Protein (APP) by Beta-site of APP Cleaving Enzyme 1 (BACE1), was conducted in this study. Briefly, HEK293 cells were colipofected with one of in total 1357 siRNAs against 60% of the human kinome and either an APP construct with only the β-cleavage site left or normally cleavable APP as control. Remaining β-cleavage was for logistic reasons firstly measured with an activity-test for secreted alkaline phosphatase (SEAP) fused to both types of APP and subjected to Aβ-ELISA when interesting. Before the screen, the APP-constructs were characterized in the cell types HEK293 and CGCs with regards to cleavage, especially by BACE1. The screen resulted in 38 hits of which one, Testis Specific Serine Kinase 3, was confirmed once more. In a second, bioinformatic project, an initially suspected APLP-like pseudogenic-like sequence in C3orf52 was refuted. Further, analysis of C3orf52 gene expression data hints on a role in myeloid leukemia. Lastly, the phylogenetic relationship of the APP family paralogs was examined, also in comparison to neighboring gene families, and found in the topology (APLP1)(APLP2/APP).
    [Show full text]
  • Open Moore Sarah P53network.Pdf
    THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY A SYSTEMATIC METHOD FOR ANALYZING STIMULUS-DEPENDENT ACTIVATION OF THE p53 TRANSCRIPTION NETWORK SARAH L. MOORE SPRING 2013 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biochemistry and Molecular Biology with honors in Biochemistry and Molecular Biology Reviewed and approved* by the following: Dr. Yanming Wang Associate Professor of Biochemistry and Molecular Biology Thesis Supervisor Dr. Ming Tien Professor of Biochemistry and Molecular Biology Honors Advisor Dr. Scott Selleck Professor and Head, Department of Biochemistry and Molecular Biology * Signatures are on file in the Schreyer Honors College. i ABSTRACT The p53 protein responds to cellular stress, like DNA damage and nutrient depravation, by activating cell-cycle arrest, initiating apoptosis, or triggering autophagy (i.e., self eating). p53 also regulates a range of physiological functions, such as immune and inflammatory responses, metabolism, and cell motility. These diverse roles create the need for developing systematic methods to analyze which p53 pathways will be triggered or inhibited under certain conditions. To determine the expression patterns of p53 modifiers and target genes in response to various stresses, an extensive literature review was conducted to compile a quantitative reverse transcription polymerase chain reaction (qRT-PCR) primer library consisting of 350 genes involved in apoptosis, immune and inflammatory responses, metabolism, cell cycle control, autophagy, motility, DNA repair, and differentiation as part of the p53 network. Using this library, qRT-PCR was performed in cells with inducible p53 over-expression, DNA-damage, cancer drug treatment, serum starvation, and serum stimulation.
    [Show full text]