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2.2.3 Generation of Genomic DNA Sequences from Plant Sources

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2.2.3 Generation of Genomic DNA Sequences from Plant Sources Copyright Undertaking This thesis is protected by copyright, with all rights reserved. By reading and using the thesis, the reader understands and agrees to the following terms: 1. The reader will abide by the rules and legal ordinances governing copyright regarding the use of the thesis. 2. The reader will use the thesis for the purpose of research or private study only and not for distribution or further reproduction or any other purpose. 3. The reader agrees to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage. IMPORTANT If you have reasons to believe that any materials in this thesis are deemed not suitable to be distributed in this form, or a copyright owner having difficulty with the material being included in our database, please contact [email protected] providing details. The Library will look into your claim and consider taking remedial action upon receipt of the written requests. Pao Yue-kong Library, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong http://www.lib.polyu.edu.hk The Hong Kong Polytechnic University Department of Health Technology and Informatics Isolation and Characterisation of Nucleases from Various Biological Sources for Further Application in Nucleic Acid Analysis By CHEUNG Tsz Shan A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy March 2012 CERTIFICATE OF ORIGINALITY I hereby declare that this thesis is my own work and that, to the best of my knowledge and belief, it reproduces no materials previously published or written, nor material that has been accepted for the award of any other degree or diploma, except where due acknowledgment has been made in the text. ________________________ (Signed) _CHEUNG Tsz Shan_______ (Name of student) I ABSTRACT Nucleases are widely used in various areas including the study of nucleic acids biochemistry, therapeutics and industrial applications. They are used in detecting polymorphisms and disease-causing mutations in nucleic acid sequences, and also used as antiviral agents or other therapeutics. More than 30 different single-strand- specific nucleases have been isolated from various sources including bacteria, fungi, plants and animals. However, only a few of them are suitable for use in research and diagnostic work. In addition, little sequence information is known about many plant enzymes that possess nuclease I characteristics. In particular, the biological role of this kind of nucleases is unclear and there are very few studies reporting the efficiencies of DNA cleavage by such nucleases. In this study, a novel single-strand-specific nuclease was isolated and functionally characterised. Eleven different plants were screened in this study. Seven out of eleven samples were found to possess the target nuclease gene that was homologous to S1 nuclease. Four novel nuclease sequences were identified from Radermachera sinica, Brassica chinesis, Brassica parachinesis and Apium graveolens var.dulce. Sequence identities, homology studies and domain/motif predictions of these sequences were characterised and determined in this study. A full-length complementary DNA (882 bp) was isolated from a potted plant, Radermachera sinica (China doll), and its protein product named as CDENDO1. Recombinant CDENDO1 protein (277 amino acid residues) was successfully cloned and purified in E. coli, and functionally characterised in this study. Recombinant CDENDO1 protein carried the S1/P1 nuclease domain characteristic of endonuclease activity and nucleic acid binding, and was classified as a member of the S1/P1 nuclease family. Recombinant CDENDO1 protein was found to behave as a single-strand-specific nuclease that was capable of hydrolysing both of single-stranded RNA and double- stranded DNA endonucleolytically. Its activity was triggered by both Ca2+/Mg2+ and inhibited by Zn2+ /Mn2+. Recombinant CDENDO1 nuclease could sustain in reaction mixtures containing low concentration of beta-mercaptoethanol and dithiothreitol. The nuclease activity was totally inactivated by either heating at 80°C or 85°C for 20 minutes, or in the presence of 20 mM EDTA. Modifications or removal of the II signal peptide of CDENDO1 nuclease did not affect the catalytic activity of CDENDO nuclease. Removal of the last twelve amino acids, i.e. G D S Q D D S L V A A T, at the C-terminal of the mature protein was found to enhance the protein expression. In this study, CDENDO1 nuclease and endonuclease V (Endo V) were first reported to be capable of cleaving the linker regions between nucleosome cores on chromatin in vitro. They might be involved in the programmed cell death pathway. Recombinant CDENDO1 nuclease were different from CEL I nuclease and S1 nuclease although they belonged to the same superfamily of S1/P1 nuclease. Although recombinant CDENDO1 nuclease failed to recognise single-nucleotide mismatch, it was capable of nicking at the linker DNA between nucleosomes on chromatin structure. This feature enables its potential application in chromatin studies. III LIST OF PUBLISHED AND PRESENTED WORK FROM THIS THESIS Conference Papers Cheung TS, Leung HM Polly, Yip SP. Nucleic acid analysis and characterisation of cloned single-strand-specific nuclease from a plant (Radermachera sinica). Proteomlux 2008: International Conference on Proteomics in Plants, Microorganisms and Environment. Oct 22-24 2008, Luxembourg. IV ACKNOWLEDGEMENTS I would like to express my gratitude to my chief supervisor, Prof. Shea Ping Yip, for his patience and advice throughout the whole PhD study. I deeply appreciate his knowledge and his assistance in report writing; and financial support during the extension period. I would like to thank other members of our department (Department of Health Technology and Informatics), Dr. Polly Leung, Dr. Tony To and Prof. Benjamin Yung, for their valuable advice, resource support and encouragement during the frustrating and harsh moments of the study. I would like to thank Dr. Larry Chow from Department of Applied Biology and Chemistry Technology, The Hong Kong Polytechnic University (PolyU), for generously providing yeast strains and vectors in the protein expression study. I would like to thank Prof. Chi-ho To and Mr. K.K. Li from School of Optometry, PolyU, for providing mass-spectrometric facilities and helping in identifying the protein products for this project. I would like to give my thanks to Miss Sharon Ho, technician from our department for helping me set up the radioactive laboratory and give me guidance in handling radioactive substance. I would like to express my sincere thanks to my friend, Dr. SW Choi, for unconditional help in editing my thesis and giving valuable comments on my writing and presentations. Grateful thanks also go to my team members and technicians from my department, for their support and kind help in collecting blood, and providing and cleaning glassware. Without their support, this work could not be run smoothly. Last but not least, I would like to express my deepest thanks to my beloved parents and friends, especially Mr. Patrick Yu, for their encouragement and unlimited support. V TABLE OF CONTENTS CERTIFICATE OF ORIGINALITY............................................................................ I ABSTRACT ......................................................................................................... II LIST OF PUBLISHED AND PRESENTED WORK FROM THIS THESIS ..............IV ACKNOWLEDGEMENTS.......................................................................................V TABLE OF CONTENTS.........................................................................................VI LIST OF FIGURES ............................................................................................. XIV LIST OF TABLES ............................................................................................. XVIII LIST OF ABBREVIATIONS................................................................................. XX Chapter 1: Introduction .................................................................................... 1 1.1 What is SNP? Significance of identifying disease caused by SNP........... 1 1.2 What are nucleases?............................................................................... 3 1.2.1 Classification of nucleases............................................................... 5 1.2.1.1 Plant nuclease.............................................................................. 7 1.2.1.2 Single-strand-specific nucleases in plants.................................... 9 1.2.1.2.1 Mung bean nuclease.............................................................. 9 1.2.1.2.2 Bifunctional nuclease, BFN1 .................................................. 9 1.2.1.2.3 Nuclease I from spinach - SP I............................................... 9 1.2.1.2.4 Nuclease I from celery - CEL I.............................................. 10 1.2.1.2.5 Nucleases from other sources.............................................. 10 1.2.1.2.6 Summary ............................................................................. 11 1.2.2 Cleavage mechanism of single-strand-specific nucleases.............. 11 1.2.3 Biological roles............................................................................... 16 1.2.4 Summary ....................................................................................... 17 1.3 Applications of nucleases .....................................................................
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