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Characterization of Cathepsin L Genes and Their Cdnas in the Brine Shrimp, Artemia Franciscana

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Characterization of Cathepsin L Genes and Their Cdnas in the Brine Shrimp, Artemia Franciscana University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 2006 Characterization of cathepsin L genes and their cDNAs in the brine shrimp, Artemia franciscana. Jian Ping Cao University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Cao, Jian Ping, "Characterization of cathepsin L genes and their cDNAs in the brine shrimp, Artemia franciscana." (2006). Electronic Theses and Dissertations. 1398. https://scholar.uwindsor.ca/etd/1398 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered. Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email ([email protected]) or by telephone at 519-253-3000ext. 3208. CHARACTERIZATION OF CATHEPSIN L GENES AND THEIR cDNAs IN THE BRINE SHRIMP, ARTEMIA FRANCISCANA By: JianPing Cao A Thesis Submitted to the Faculty of Graduate Studies and Research through the Department of Biological Sciences in Partial Fulfillment of the Requirements for the Degree of Master of Science at the University of Windsor Windsor, Ontario, Canada 2006 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Library and Bibliotheque et Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-17101-1 Our file Notre reference ISBN: 978-0-494-17101-1 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce,Canada de reproduire, publier, archiver, publish, archive, preserve, conserve,sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet,distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform,et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be includedBien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. i * i Canada Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cao JianPing Copyright© 2006 All rights reserved Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT: Embryos of the brine shrimp,Artemia franciscana, contain a novel cysteine protease composed of a cathepsin L-Iike catalytic subunit of 28.5 kDa, and a cell adhesion protein of the FAS-1 family of 31.5 kDa. The cathepsin L-like subunit is encoded by a cDNA derived fromCL-1 the gene inA. franciscana which has been shown to be intron-less. TheCL-1 gene was detected in genomic DNA prepared from nuclei of Artemia, and a secondCL gene (CL-2 ) was obtained from a genomic library in EMBL3. Screeningo f Artemia adult and embryo cDNA libraries yielded sequences homologous to the CL-2 gene, and confirmed thatCL-2 gene is also intron-less. Artemia adult CL-2 cDNA was found to contain two distinct open reading frames encoding the pro-peptide and mature protease. Several potential transcription factor binding sites were identified, indicating the possibility of a functional promoter in the 5’ upstream sequence of the CL-2 gene. iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dedicated to Dr. A. H. Warner V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENT I wish to thank my supervisor Dr. A. H. Warner very much for providing me the opportunity to work in his laboratory as a Master’s student. His advice and training during this period are very much appreciated. I would also like to thank my committee member Dr. J. Hudson from the Department of Biological Sciences and Dr. B. Mutus from the department of Chemistry and Biochemistry for their time to review my thesis. Many thanks are also due to the graduate students and the technical support staff of this department for their help. vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ABSTRACT....................................................................................................iv DEDICATION................................................................................................v ACKNOWLEDGEMENT...........................................................................vi LIST OF FIGURES...................................................................................... xi INTRODUCTION......................................................................................... 1 1. Artemia .........................................................................................................1 2. Cysteine protease....................................................................................... 2 3. Papain family.............................................................................................. 2 4. Cathepsin L.................................................................................................5 5. Pro-region of cathepsin L..........................................................................7 6. Intracellular protease targeting.................................................................7 6.1 Mannose-6-phosphate dependent process.......................................7 6.2 Mannose-6-phosphate independent process....................................8 7. Human cathepsin Lgene.......................................................................... 8 8. Cathepsin Lgenes of the shrimp Penaeus vannamei............................9 9. Cathepsin Z,-like cysteine proteases gene of Leishmania............................................................................................. 9 10.Cathepsin Lgene ofFasciola gigantica..................................................10 11 .Artemia franciscana cathepsin L..............................................................11 12.Cysteine protease inhibitors.......................................................................12 13. Objectives..................................................................................................... 15 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M A TERIALS..................................................................................................16 M ETH O D S......................................................................................................17 1. Isolation of cDNA clones coding forArtemia franciscana embryo cathepsin L..................................................................................... 17 2. Construction of [32p]-labeled CL cDNA probe.....................................18 3. Purification of PCR products....................................................................18 4. Cloning of PCR products.......................................................................... 19 4.1 DNA ligation reaction.......................................................................19 4.2 Transformation of competentEscherichia coli cells....................19 5. Characterization ofArtemia cathepsin L-l gene isolated from genomic DNA.................................................................................. 20 5.1 Isolation ofArtemia nuclei.............................................................. 20 5.2 Isolation ofArtemia franciscana genomic DNA...........................20 6. Screening of recombinant DNA clones forArtemia franciscana cathepsin L nucleotide sequences........................................................... 21 7. Isolation of plasmid DNA from CL positiveArtemia franciscana clones............................................................................................................21
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