DOCSLIB.ORG

Escherichia Coli

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Escherichia Coli Kent Academic Repository Full text document (pdf) Citation for published version Malherbe, Gilles (2019) Evaluation of the Tat export pathway for the production of recombinant proteins in Escherichia coli. Doctor of Philosophy (PhD) thesis, University of Kent,. DOI Link to record in KAR https://kar.kent.ac.uk/81263/ Document Version UNSPECIFIED Copyright & reuse Content in the Kent Academic Repository is made available for research purposes. Unless otherwise stated all content is protected by copyright and in the absence of an open licence (eg Creative Commons), permissions for further reuse of content should be sought from the publisher, author or other copyright holder. Versions of research The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record. Enquiries For any further enquiries regarding the licence status of this document, please contact: [email protected] If you believe this document infringes copyright then please contact the KAR admin team with the take-down information provided at http://kar.kent.ac.uk/contact.html Evaluation of the Tat export pathway for the production of recombinant proteins in Escherichia coli Gilles Malherbe Thesis submitted to the University of Kent for the degree of Doctor of Philosophy in the Faculty of Microbiology 2019 ABSTRACT Recently, the twin-arginine translocation (Tat) pathway has raised interest due to reports of its unique proofreading ability to export correctly folded proteins. However, only a few recombinant proteins fused to an N-terminal Tat signal peptide have been reported to be exported via Tat. The hypothesis for this thesis was to evaluate whether the range of proteins of interest that can be exported by the Tat pathway can be extended by C-terminally fusing them to a natural Tat substrate (NTS). Thus, the NTS would act as a soluble carrier to improve cytoplasmic solubility and facilitate Tat export. Moreover, the CyDisCo technology enabling cytoplasmic formation of disulphide bonds was investigated to reach this goal. Initially, a robust fractionation method was developed to assess protein localisation with due diligence. A panel of NTS and reporter proteins were evaluated for the respective role of the soluble carrier and the protein of interest. None of the selected NTS and only PhoA and hGH of the tested reporter proteins were shown to export specifically via Tat. After the review of the fusion design to use the valid reporter proteins as the soluble carrier, three proteins of interest were expressed with and without the soluble carriers. This fusion strategy did not extend Tat acceptance. Overall, this project confirmed the difficulty to export a recombinant protein via the Tat pathway. Additionally, CyDisCo was demonstrated to improve expression of recombinant proteins but did not form native disulphide bonds. However, this project revealed unexpected export of sfGFP, hGH and FABP4 including the recombinant proteins fused to hGH without a signal peptide. This unidentified translocation mechanism(s) needs further characterisation which can lead to the discovery of a new export pathway(s) and may highlight a promising alternative way to target proteins of interest to the periplasm. I TABLE OF CONTENT ABSTRACT ............................................................................................ I TABLE OF CONTENT ............................................................................ II LIST OF FIGURES .............................................................................. VIII LIST OF TABLES ................................................................................... X LIST OF APPENDICES .......................................................................... XI DECLARATION ................................................................................... XII ABBREVIATIONS AND SYMBOLS ...................................................... XIII ACKNOWLEDGEMENTS ................................................................... XVI 1. INTRODUCTION .............................................................................. 1 1.1. General introduction ........................................................................ 1 1.2. The Escherichia coli K12 strain as a production host ....................... 3 1.3. Secretion systems in Escherichia coli ............................................... 5 1.3.1. The Sec pathway ...................................................................................... 5 1.3.1.1. Sec-specific signal peptides ............................................................. 5 1.3.1.2. The SecB pathway ........................................................................... 8 1.3.1.3. The SRP pathway ............................................................................. 9 1.3.1.4. Recombinant protein export via Sec ............................................. 10 1.3.2. The twin arginine translocation pathway ............................................ 10 1.3.2.1. The role of Tat through its substrates ........................................... 11 1.3.2.2. Tat-specific signal peptides ........................................................... 12 1.3.2.3. Components of Tat ........................................................................ 13 1.3.2.4. Tat export mechanism ................................................................... 15 1.3.2.5. The proofreading mechanism ....................................................... 18 1.3.2.6. The Tat systems beyond E. coli ...................................................... 19 1.3.3. The Type I to VI secretion pathways .................................................... 21 1.3.4. Unknown secretion pathway(s) ............................................................ 23 1.4. Engineering to improve E. coli as an expression host .................... 25 1.4.1. Disulphide bond formation ................................................................... 25 II 1.4.1.1. The naturally oxidising periplasmic space ..................................... 26 1.4.1.2. Disruption of the reducing pathways ............................................ 26 1.4.1.3. The CyDisCo technology ................................................................ 27 1.4.2. Glycosylation in E. coli ........................................................................... 28 1.4.3. Codon usage .......................................................................................... 29 1.4.4. Stabilization of the recombinant product ............................................ 29 1.4.4.1. Use of protease-deficient strains .................................................. 30 1.4.4.2. Co-expressing chaperones ............................................................ 30 1.4.4.3. Fusion of a soluble partner ............................................................ 31 1.4.4.4. Improving the process ................................................................... 31 1.4.5. Other engineered strains ...................................................................... 31 1.5. Aims and hypothesis ...................................................................... 32 2. MATERIALS AND METHODS ......................................................... 34 2.1. Strains ............................................................................................. 34 2.2. Molecular biology ........................................................................... 35 2.2.1. Transformation of chemically competent bacteria by heat shock ..... 35 2.2.2. Plasmid amplification ............................................................................ 35 2.2.3. Amplification by PCR ............................................................................. 35 2.2.4. PCR purification ..................................................................................... 36 2.2.5. DNA digestion with restriction enzymes .............................................. 36 2.2.6. Agarose gel electrophoresis.................................................................. 36 2.2.7. Agarose gel extraction .......................................................................... 36 2.2.8. Ligation ................................................................................................... 37 2.2.9. Directed mutagenesis ........................................................................... 37 2.2.10. Type IIS cloning strategy ..................................................................... 37 2.2.10.1. Design of backbone vector .......................................................... 37 2.2.10.2. Design of inserts .......................................................................... 38 2.2.10.3. Type IIS cloning reaction ............................................................. 38 2.2.11. Plasmid DNA sequencing .................................................................... 39 2.2.12. Genomic DNA extraction .................................................................... 42 2.2.13. Genomic DNA sequencing .................................................................. 43 2.2.14. Total RNA extraction ........................................................................... 43 III 2.2.15. Reverse transcription .......................................................................... 43 2.3. Protein expression .........................................................................
Load more

Recommended publications
  • California Marine Life Protection Act (MLPA) Initiative
    California Marine Life Protection Act (MLPA) Initiative Evaluation of Existing Central Coast Marine Protected Areas DRAFT, v. 2.0 November 4, 2005 CONTENTS EXECUTIVE SUMMARY ..........................................................................................................................4 1.0 INTRODUCTION ................................................................................................................................8 2.0 EVALUATION OF EXISTING MPAs .................................................................................................11 2.1 Año Nuevo Special Closure...........................................................................................................11 2.2 Elkhorn Slough State Marine Reserve ...........................................................................................12 2.3 Hopkins State Marine Reserve......................................................................................................15 2.4 Pacific Grove State Marine Conservation Area.............................................................................16 2.5: Carmel Bay State Marine Conservation Area ..............................................................................17 2.6 Point Lobos State Marine Reserve................................................................................................19 2.7 Julia Pfeiffer Burns State Marine Conservation Area.....................................................................20 2.8 Big Creek State Marine Reserve....................................................................................................22
    [Show full text]
  • Ereskovsky Et 2017 Oscarella S
    Transcriptome sequencing and delimitation of sympatric Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA Alexander Ereskovsky, Daniel Richter, Dennis Lavrov, Klaske Schippers, Scott Nichols To cite this version: Alexander Ereskovsky, Daniel Richter, Dennis Lavrov, Klaske Schippers, Scott Nichols. Transcriptome sequencing and delimitation of sympatric Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA. PLoS ONE, Public Library of Science, 2017, 12 (9), pp.1-25/e0183002. 10.1371/jour- nal.pone.0183002. hal-01790581 HAL Id: hal-01790581 https://hal.archives-ouvertes.fr/hal-01790581 Submitted on 14 May 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License RESEARCH ARTICLE Transcriptome sequencing and delimitation of sympatric Oscarella species (O. carmela and O. pearsei sp. nov) from California, USA Alexander V. Ereskovsky1,2, Daniel J. Richter3,4, Dennis V. Lavrov5, Klaske J. Schippers6, Scott A. Nichols6* 1 Institut MeÂditerraneÂen de BiodiversiteÂet d'Ecologie Marine et Continentale (IMBE), CNRS, IRD, Aix Marseille UniversiteÂ, Avignon UniversiteÂ, Station Marine d'Endoume, Marseille, France, 2 Department of a1111111111 Embryology, Faculty of Biology, Saint-Petersburg State University, 7/9 Universitetskaya emb., St.
    [Show full text]
  • Demospongiae) Asociadas a Comunidades Coralinas Del Pacífico Mexicano
    INSTITUTO POLITÉCNICO NACIONAL CENTRO INTERDISCIPLINARIO DE CIENCIAS MARINAS COMPOSICIÓN Y AFINIDADES BIOGEOGRÁFICAS DE ESPONJAS (DEMOSPONGIAE) ASOCIADAS A COMUNIDADES CORALINAS DEL PACÍFICO MEXICANO T E S I S QUE PARA OBTENER EL GRADO DE DOCTORADO EN CIENCIAS MARINAS P R E S E N T A CRISTINA VEGA JUÁREZ LA PAZ, B. C. S. JUNIO DEL 2012 INSTITUTO POLITÉCNICO NACIONAL SECRETARÍA DE INVESTIGACIÓN Y POSGRADO CARTA CESIÓN DE DERECHOS En la Ciudad de La Paz, B.C.S., el día 23 del mees Mayo del año 2012 el (la) que suscribe MC. CRISTINA VEGA JUÁREZ alumno(a) del Programa de DOCTORADO EN CIENCIAS MARINAS con número de registro B081062 adscrito al CENTRO INTERDISCIPLINARIO DE CIENCIAS MARINAS manifiesta que es autor (a) intelectual del presente trabajo de tesis, baajjo la dirección de: DRA. CLAUDIA JUDITH HERNÁNDEZ GUERRERO Y DR. JOSSÉ LUIS CARBALLO CENIZO y cede los derechos del trabajo titulado: “COMPOSICIÓN Y AFINIDADES BIOGEOGRÁFICAS DE ESPONJAS (DEMOSPONGIAE) ASOCIADAS A COMUNIDADES CORALINAS DEL PACÍFICO MEXICANO” al Instituto Politécnico Nacional, para su difusión con fines académicos y de investigación. Los usuarios de la información no deben reproducir el contenido textual, gráficas o datos del trabajo sin el permiso expreso del autor y/o director del trabajo. Éste, puede ser obtenido escribiendo a la Siguiente dirección: [email protected] - [email protected] [email protected] Si el permiso se otorga, el usuario deberá dar el agradecimiento correspondiente y citar la fuente del mismo. MC. CRISTINA VEGA JUÁREZ nombre y firmaa AGRADECIMIENTOS Al Instituto Politécnico Nacional a través del Centro Interdisciplinario de Ciencias Marinas, La Paz, B.C.S.
    [Show full text]
  • (Homoscleromorpha) with Description of Two New Oscarella Species
    Systematics and Molecular Phylogeny of the Family Oscarellidae (Homoscleromorpha) with Description of Two New Oscarella Species Eve Gazave, Dennis Lavrov, Jory Cabrol, Emmanuelle Renard, Caroline Rocher, Jean Vacelet, Maja Adamska, Carole Borchiellini, Alexander Ereskovsky To cite this version: Eve Gazave, Dennis Lavrov, Jory Cabrol, Emmanuelle Renard, Caroline Rocher, et al.. Systematics and Molecular Phylogeny of the Family Oscarellidae (Homoscleromorpha) with Description of Two New Oscarella Species. PLoS ONE, Public Library of Science, 2013, 160, pp.781 - 781. 10.1371/jour- nal.pone.0063976.s006. hal-01456636 HAL Id: hal-01456636 https://hal.archives-ouvertes.fr/hal-01456636 Submitted on 13 Apr 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Systematics and Molecular Phylogeny of the Family Oscarellidae (Homoscleromorpha) with Description of Two New Oscarella Species Eve Gazave1,2*, Dennis V. Lavrov3, Jory Cabrol2, Emmanuelle Renard2, Caroline Rocher2, Jean Vacelet2, Maja Adamska4, Carole Borchiellini2, Alexander V. Ereskovsky2,5 1 Institut
    [Show full text]
  • UCLA Electronic Theses and Dissertations
    UCLA UCLA Electronic Theses and Dissertations Title Comparative and developmental genomics in the moon jellyfish Aurelia species 1 Permalink https://escholarship.org/uc/item/92p1s9r5 Author Gold, David Publication Date 2014 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles Comparative and developmental genomics in the moon jellyfish Aurelia species 1 A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by David Adler Gold 2014 © Copyright by David Adler Gold 2014 ABSTRACT OF THE DISSERTATION Comparative and developmental genomics in the moon jellyfish Aurelia species 1 by David Adler Gold Doctor of Philosophy in Biology University of California, Los Angeles, 2014 Professor David K. Jacobs, Chair This dissertation focuses on the transcriptome of the moon jellyfish (Aurelia sp.1). As the chapters progress, larger sets of genes are analyzed, and the work becomes decreasingly comparative in nature, and increasingly focused on Aurelia. In the first chapter, I analyze the POU-class genes. I begin by using comparative genomics and “gene fishing” to resolve the topology of the POU gene tree. I then use ancestral state reconstruction to map the most likely changes in amino acid evolution for the conserved protein domains. Four of the six POU families evolved before the last common ancestor of living animals—doubling previous estimates—and was followed by extensive clade-specific gene loss. POU families best understood for their generic roles in cell-type regulation and stem cell pluripotency (POU2, POU5) show the largest number of nonsynonymous mutations, suggestive of functional evolution, while those better known for specifying subsets of neural and hormone- producing cell types (POU1, POU3) appear more similar to the ancestral protein.
    [Show full text]
  • The Evolution of the Mitochondrial Genomes of Calcareous Sponges and Cnidarians Ehsan Kayal Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 The evolution of the mitochondrial genomes of calcareous sponges and cnidarians Ehsan Kayal Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Evolution Commons, and the Molecular Biology Commons Recommended Citation Kayal, Ehsan, "The ve olution of the mitochondrial genomes of calcareous sponges and cnidarians" (2012). Graduate Theses and Dissertations. 12621. https://lib.dr.iastate.edu/etd/12621 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The evolution of the mitochondrial genomes of calcareous sponges and cnidarians by Ehsan Kayal A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Ecology and Evolutionary Biology Program of Study Committee Dennis V. Lavrov, Major Professor Anne Bronikowski John Downing Eric Henderson Stephan Q. Schneider Jeanne M. Serb Iowa State University Ames, Iowa 2012 Copyright 2012, Ehsan Kayal ii TABLE OF CONTENTS ABSTRACT ..........................................................................................................................................
    [Show full text]
  • View=TSA&Search=Halisarca Adapter Index Sequence Before Sequences Were Uploaded to Their Server
    Marine Genomics 37 (2018) 135–147 Contents lists available at ScienceDirect Marine Genomics journal homepage: www.elsevier.com/locate/margen Towards the identification of ancestrally shared regenerative mechanisms T across the Metazoa: A Transcriptomic case study in the Demosponge Halisarca caerulea Nathan J. Kennya,1, Jasper M. de Goeijb,1, Didier M. de Bakkerb, Casey G. Whalenb, ⁎ Eugene Berezikovc, Ana Riesgoa, a Life Sciences Department, The Natural History Museum, Cromwell Road, London SW7 5BD, UK b Department of Freshwater and Marine Science, University of Amsterdam, Postbus 94240, 1090 GE, Amsterdam, The Netherlands c European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands ARTICLE INFO ABSTRACT Keywords: Regeneration is an essential process for all multicellular organisms, allowing them to recover effectively from Regeneration internal and external injury. This process has been studied extensively in a medical context in vertebrates, with Transcriptome pathways often investigated mechanistically, both to derive increased understanding and as potential drug Halisarca caerulea, Porifera, ancestral cassette targets for therapy. Several species from other parts of the metazoan tree of life, including Hydra, planarians and echinoderms, noted for their regenerative capabilities, have previously been targeted for study. Less well- documented for their regenerative abilities are sponges. This is surprising, as they are both one of the earliest- branching extant metazoan phyla on Earth, and are rapidly able to respond to injury. Their sessile lifestyle, lack of an external protective layer, inability to respond to predation and filter-feeding strategy all mean that re- generation is often required. In particular the demosponge genus Halisarca has been noted for its fast cell turnover and ability to quickly adjust its cell kinetic properties to repair damage through regeneration.
    [Show full text]
  • Origin of Metazoan Cadherin Diversity and the Antiquity of the Classical Cadherin/Β-Catenin Complex
    Origin of metazoan cadherin diversity and the antiquity of the classical cadherin/β-catenin complex Scott Anthony Nicholsa, Brock William Robertsb, Daniel Joseph Richterb, Stephen Robert Faircloughb, and Nicole Kingb,1 aDepartment of Biological Sciences, University of Denver, Denver, CO 80208; and bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Edited by Masatoshi Takeichi, RIKEN, Kobe, Japan, and approved June 20, 2012 (received for review December 19, 2011) The evolution of cadherins, which are essential for metazoan multi- flamingo, dachsous, fat, and fat-like cadherins regulate planar cellularity and restricted to metazoans and their closest relatives, cell polarity in organisms as disparate as Drosophila and mouse has special relevance for understanding metazoan origins. To re- (20–22). Members of the protocadherin family have diverse construct the ancestry and evolution of cadherin gene families, we functions that include mechanosensation in stereocilia and reg- analyzed the genomes of the choanoflagellate Salpingoeca rosetta, ulation of nervous system development (23, 24). It is not known the unicellular outgroup of choanoflagellates and metazoans Cap- whether the bilaterian roles of these cadherin families had al- saspora owczarzaki, and a draft genome assembly from the homo- ready evolved in the last common ancestor of metazoans, and it scleromorph sponge Oscarella carmela. Our finding of a cadherin is not clear how these cadherin families themselves originated. gene in C. owczarzaki reveals that cadherins predate the diver- To date, only one cadherin family—the hedgling family—is gence of the C. owczarzaki, choanoflagellate, and metazoan line- inferred to have been present in the last common ancestor of ages.
    [Show full text]
  • Exploring the Bzip Regulatory Network in the Sponge Amphimedon Queenslandica: Insights Into the Ancestral Animal Regulatory Genome
    Exploring the bZIP regulatory network in the sponge Amphimedon queenslandica: Insights into the ancestral animal regulatory genome Katia Jindrich B.Sc, M.Sc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Biological Sciences I Abstract What genomic innovations supported the emergence of multicellular animals, over 600 million years ago, remains one of the most fundamental questions in evolutionary biology. Increasing evidence suggests that the elaboration of the regulatory mechanisms controlling gene expression, rather than gene innovation, underlies this transition. Since transcriptional regulation is largely achieved through the binding of specific transcription factors to specific cis-regulatory DNA, understanding the early evolution of these master orchestrators is key to retracing the origin of animals (metazoans). Basic leucine zipper (bZIP) transcription factors constitute one of the most ancient and conserved families of transcriptional regulators. They play a pivotal role in multiple pathways that regulate cell decisions and behaviours in all kingdoms of life. Here, I explore the early evolution and putative roles of bZIPs in a representative of one of the oldest surviving animal phyla, the marine sponge Amphimedon queenslandica. Using recently sequenced genomes from across the Eukaryota, and in particular the Metazoa, I first reconstruct the evolution of bZIPs, and document that this transcription factor superfamily has undergone multiple independent kingdom-level expansions. I present here a thorough analysis of the whole superfamily of bZIP transcription factors across the main eukaryotic clades and the putative bZIP network that was in place in the ancestor of all extant animals. To then explore the putative role of bZIP transcription factors in the metazoan ancestor, I identify the bZIP complement in the demosponge Amphimedon queenslandica (phylum Porifera).
    [Show full text]
  • Six Major Steps in Animal Evolution: Are We Derived Sponge Larvae?
    EVOLUTION & DEVELOPMENT 10:2, 241–257 (2008) Six major steps in animal evolution: are we derived sponge larvae? Claus Nielsen Zoological Museum (The Natural History Museum of Denmark, University of Copenhagen), Universitetsparken 15, DK-2100 Copenhagen, Denmark Correspondence (email: [email protected]) SUMMARY A review of the old and new literature on animal became sexually mature, and the adult sponge-stage was morphology/embryology and molecular studies has led me to abandoned in an extreme progenesis. This eumetazoan the following scenario for the early evolution of the metazoans. ancestor, ‘‘gastraea,’’ corresponds to Haeckel’s gastraea. The metazoan ancestor, ‘‘choanoblastaea,’’ was a pelagic Trichoplax represents this stage, but with the blastopore spread sphere consisting of choanocytes. The evolution of multicellularity out so that the endoderm has become the underside of the enabled division of labor between cells, and an ‘‘advanced creeping animal. Another lineage developed a nervous system; choanoblastaea’’ consisted of choanocytes and nonfeeding cells. this ‘‘neurogastraea’’ is the ancestor of the Neuralia. Cnidarians Polarity became established, and an adult, sessile stage have retained this organization, whereas the Triploblastica developed. Choanocytes of the upper side became arranged in (Ctenophora1Bilateria), have developed the mesoderm. The a groove with the cilia pumping water along the groove. Cells bilaterians developed bilaterality in a primitive form in the overarched the groove so that a choanocyte chamber was Acoelomorpha and in an advanced form with tubular gut and formed, establishing the body plan of an adult sponge; the pelagic long Hox cluster in the Eubilateria (Protostomia1Deuterostomia). larval stage was retained but became lecithotrophic. The It is indicated that the major evolutionary steps are the result of sponges radiated into monophyletic Silicea, Calcarea, and suites of existing genes becoming co-opted into new networks Homoscleromorpha.
    [Show full text]
  • Calcareous Sponge Genomes Reveal Complex Evolution of A-Carbonic
    Calcareous sponge genomes reveal complex evolution of α-carbonic anhydrases and two key biomineralization enzymes Voigt et al. Voigt et al. BMC Evolutionary Biology 2014, 14:230 http://www.biomedcentral.com/1471-2148/14/230 Voigt et al. BMC Evolutionary Biology 2014, 14:230 http://www.biomedcentral.com/1471-2148/14/230 RESEARCH ARTICLE Open Access Calcareous sponge genomes reveal complex evolution of α-carbonic anhydrases and two key biomineralization enzymes Oliver Voigt1*, Marcin Adamski2, Kasia Sluzek1 and Maja Adamska2* Abstract Background: Calcium carbonate biominerals form often complex and beautiful skeletal elements, including coral exoskeletons and mollusc shells. Although the ability to generate these carbonate structures was apparently gained independently during animal evolution, it sometimes involves the same gene families. One of the best-studied of these gene families comprises the α- carbonic anhydrases (CAs), which catalyse the reversible transformation of CO2 − to HCO3 and fulfill many physiological functions. Among Porifera –the oldest animal phylum with the ability to produce skeletal elements– only the class of calcareous sponges can build calcitic spicules, which are the extracellular products of specialized cells, the sclerocytes. Little is known about the molecular mechanisms of their synthesis, but inhibition studies suggest an essential role of CAs. In order to gain insight into the evolution and function of CAs in biomineralization of a basal metazoan species, we determined the diversity and expression of CAs in the calcareous sponges Sycon ciliatum and Leucosolenia complicata by means of genomic screening, RNA-Seq and RNA in situ hybridization expression analysis. Active biomineralization was located with calcein-staining. Results: We found that the CA repertoires of two calcareous sponge species are strikingly more complex than those of other sponges.
    [Show full text]
  • Examples from Non-Bilaterian Animals" (2014)
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 The mitochondrial ratchet: Examples from non- bilaterian animals Walker Pett Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Bioinformatics Commons Recommended Citation Pett, Walker, "The mitochondrial ratchet: Examples from non-bilaterian animals" (2014). Graduate Theses and Dissertations. 14035. https://lib.dr.iastate.edu/etd/14035 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The mitochondrial ratchet: Examples from non-bilaterian animals by Walker Pett A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Bioinformatics and Computational Biology Program of Study Committee Dennis V. Lavrov, Co-major Professor Karin Dorman, Co-major Professor Jonathan Wendel Jeanne Serb Amy Andreotti Iowa State University Ames, Iowa 2014 ii Each of my nematocyst limbs shift and pulse on ocean, dangling in salt for tiny meals of cytoplasmic meat. Your gills and silvered skins still microscopic, your uniformed fragile bodies catch against my tentacle-teeth as I weave through depths, past pelagic zones to trail like finely threaded angry lace, down where light has long been filtered out, diluted. I swing through you, clouds of flagellates, you unholy vulnerable masses of genetic matter, scattered across my currents, forgotten by those who left you, equal, unnamed.
    [Show full text]