by Chan Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 120-137). / Acknowledgments --- p.i / Table of Content --- p.ii / List of Tables --- p.vi / List of Figures --- p.viii / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- Literature Review Page / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- "Overview of the Structures of GH, PRL and SL" --- p.2 / Chapter 1.3 --- Physiological Significance of the Hormones in Teleost --- p.12 / Chapter 1.3.1 --- Growth Hormone --- p.12 / Chapter 1.3.2 --- Prolactin --- p.12 / Chapter 1.3.3 --- Somatolactin --- p.13 / Chapter 1.4 --- Structure - Function Relationship and Receptor Binding Activities --- p.15 / Chapter 1.5 --- Aims of Thesis --- p.23 / Chapter CHAPTER 2 --- General Methodology / Chapter 2.1 --- Materials --- p.25 / Chapter 2.2 --- DNA manipulation methods --- p.28 / Chapter 2.2.1 --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.2.2 --- Ethanol Precipitation of DNA --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis of DNA --- p.29 / Chapter 2.2.4 --- Sephadex G-50 Spun Column --- p.30 / Chapter 2.2.5 --- Nick Translation --- p.30 / Chapter 2.2.6 --- Small Scale Plasmid Preparation by Alkaline Lysis Method --- p.31 / Chapter 2.2.7 --- Large Scale Plasmid Preparation Using Magic´ёØ Maxipreps DNA Purification System (Promega) --- p.32 / Chapter 2.3 --- DNA Cloning Methods --- p.33 / Chapter 2.3.1 --- Blunt-end Ligation of PCR Product to pUC18 Vector --- p.33 / Chapter 2.3.1.1 --- Preparation and Transformation of E. coli (JM109) Competent Cell --- p.33 / Chapter 2.3.1.2 --- Recovery of DNA from Agarose Gel Using the Sephaglas Bandprep Kit (Pharmacia Biotech) --- p.34 / Chapter 2.3.1.3 --- Kinasing Reaction --- p.34 / Chapter 2.3.1.4 --- Klenow Fill-in Reaction and Ligation of DNA Fragments --- p.35 / Chapter 2.3.2 --- Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.1 --- Preparation of Host Cell for Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.2 --- Phage Stock Tittering --- p.36 / Chapter 2.3.2.3 --- Plaque Lifting and Fixation on Nylon Membranes --- p.36 / Chapter 2.3.2.4 --- Library Screening by Hybridization --- p.37 / Chapter 2.3.2.5 --- In vivo Excision --- p.37 / Chapter 2.4 --- Nucleotide Sequence Determination using Dideoxy Nucleotide Chain Termination Method --- p.38 / Chapter 2.5 --- Protein methods --- p.39 / Chapter 2.5.1 --- Bicinchoninic Acid (BCA) Assay --- p.39 / Chapter 2.5.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter CHAPTER 3 --- Isolation and Characterization of cDNA Clones for Goldfish Prolactin / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Preparation of Hormone Specific DNA Probes by PCR Cloning --- p.43 / Chapter 3.2.2 --- Construction of Goldfish Pituitary cDNA Library --- p.44 / Chapter 3.2.3 --- cDNA Library Screening for Hormone Specific cDNAs --- p.44 / Chapter 3.2.4 --- Restriction Enzyme Digestion of the cDNA Clones and Subcloning of the Restriction Fragments --- p.45 / Chapter 3.2.5 --- Nucleotide Sequences Determination using Dideoxy Nucleotide Chain Termination Method --- p.46 / Chapter 3.2.6 --- Southern Analysis of the Goldfish Genomic DNA --- p.47 / Chapter 3.2.7 --- Northern Blot Analyses of Goldfish Pituitary Total RNA --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Screening of the Library and Characterization of the Clones --- p.49 / Chapter 3.3.1.1 --- Analyses of the cDNA Clones --- p.49 / Chapter 3.3.1.2 --- Cross Hybridization Reactivity --- p.57 / Chapter 3.3.2 --- Genomic Southern Blot Analyses --- p.58 / Chapter 3.3.3 --- Gene Expression of the Pituitary Hormones - Northern Blot Analyses --- p.61 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- Cross Hybridization Reavtivities of the Probes to the Hormone cDNA Clones --- p.64 / Chapter 3.4.2 --- Structural Analyses of the Hormone cDNA Clones --- p.65 / Chapter 3.4.2.1 --- Growth Hormone --- p.65 / Chapter 3.4.2.2 --- Prolactin --- p.68 / Chapter 3.4.2.3 --- Somatolactin --- p.70 / Chapter 3.4.3 --- Secondary Structure Prediction --- p.72 / Chapter 3.4.4 --- Genomic Southern Analyses --- p.78 / Chapter 3.4.5 --- Pituitary Expression of Goldfish Hormone mRNAs --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter CHAPTER 4 --- "Expression of Recombinant Goldfish Growth Hormone, Prolactin and Somatolactin in Escherichia coli (E. coli)" / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and Methods --- p.83 / Chapter 4.2.1 --- Materials --- p.83 / Chapter 4.2.2 --- Construction of Expression Vectors Carrying the Hormone Coding Regions --- p.84 / Chapter 4.2.3 --- Pilot Expression Experiment to Determine Kinetics of Expression --- p.86 / Chapter 4.2.4 --- "Large Scale Expression and Purification of Recombinant Hormones by Ni2+-NTA Affinity Column (ProBond´ёØ column, Invitrogen)" --- p.86 / Chapter 4.2.5 --- Western Blotting of the Recombinant Hormones on Polyvinylidene Fluoride (PVDF) Membrane --- p.87 / Chapter 4.2.6 --- Dot Blot Immobilisation of the Recombinant Hormones on Nitrocellulose Membrane --- p.88 / Chapter 4.2.7 --- Detection of the Blotted Protein by Enzyme-linked Immunodetaction Method --- p.88 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Construction of the Expression Vectors --- p.90 / Chapter 4.3.2 --- Expression of the Recombinant Hormones --- p.97 / Chapter 4.3.3 --- Partial Purification and Analyses of the recombinant Hormones --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Construction of the Expression Vectors --- p.105 / Chapter 4.4.2 --- Expression of the Recombinant Hormones --- p.108 / Chapter 4.4.3 --- Partial Purification and Characterization of the Recombinant Hormones --- p.109 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- General Discussion --- p.115 / References --- p.120
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321578 |
| Date | January 1996 |
| Contributors | Chan, Yuk Hang., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiii, 137 leaves : ill. (some col., some mounted) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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